System and method for diagnosis and treatment

ABSTRACT

This invention relates the use of cortisol blockers (glucocorticoid receptor [GR] antagonists) for the treating or preventing treatment resistant prostate cancer, treating or preventing neoplasia, and treating or preventing infection related to acute or chronic injury or disease.

This application is a Continuation application of U.S. Ser. No.16/251,299 filed Jan. 18, 2019, which is a Continuation application ofU.S. Ser. No. 16/050,155 filed Jul. 31, 2018, now U.S. Pat. No.10,231,982 issued Mar. 19, 2019, which is a Divisional application ofU.S. Ser. No. 15/854,053 filed Dec. 26, 2017, now U.S. Pat. No.10,076,528, issued Sep. 18, 2018, which is a Divisional application ofU.S. Ser. No. 15/471,123, filed Mar. 28, 2017, now U.S. Pat. No.9,861,643 issued Jan. 9, 2018, which is a Divisional application of U.S.Ser. No. 15/095,293, filed Apr. 11, 2016, now U.S. Pat. No. 9,636,351issued May 2, 2017, which is a Divisional application of U.S. Ser. No.14/802,060 filed Jul. 17, 2015, now U.S. Pat. No. 9,314,473, issued Apr.19, 2016, which is a Continuation-in-Part of U.S. Ser. No. 14/100,714filed Dec. 9, 2013, now U.S. Pat. No. 9,114,117, issued Aug. 25, 2015,which is a Divisional application of U.S. Ser. No. 13/364,651, filedFeb. 2, 2012, now U.S. Pat. No. 8,658,128, issued Feb. 25, 2014, whichclaims benefit under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication 61/462,492 filed Feb. 3, 2011; U.S. Provisional PatentApplication 61/463,212 filed Feb. 14, 2011; U.S. Provisional PatentApplication 61/465,703 filed Mar. 23, 2011; U.S. Provisional PatentApplication 61/518,248 filed May 3, 2011; and U.S. Provisional PatentApplication 61/519,323 filed May 20, 2011, the disclosures of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION 1. Field of Invention

This invention relates the use of cortisol blockers (e.g.,glucocorticoid receptor [GR] antagonists) for the treating or preventingtreatment resistant prostate cancer, treating or preventing neoplasia,and treating or preventing infection related to acute or chronic injuryor disease.

2. Description of Related Art

ORG 34517 is one of a class of therapeutic agents designed to block theglucorticoid receptor (GR), acting as an antagonist for endogenouscortisol. Its primary developmental pathway has been as a treatment forneuropsychiatric diseases that are characterized by dysregulatedsignaling in the hypothalamic-pituitary-adrenal axis, often with higherthan normal circulating levels of endogenous cortisol. Of particularnote are the phase 2 clinical trials that have been completed for thetreatment of psychotic depression. Other possible uses in this diseasecategory which are under investigation include: post-traumatic stressdisorder, weight gain in patients requiring long term anti-psychoticmedication, hospital delirium of the elderly, etc.

The endogenous glucocorticoids are steroids predominantly produced inthe adrenal cortex. Glucocorticoids are important steroids forintermediary metabolism, immune, musculoskeletal, connective tissue andbrain function. The main glucocorticoid in the body is cortisol. Theproduction and secretion of cortisol is governed by a complex and highlyefficient system that includes the hypothalamus, pituitary and theadrenal glands i.e., hypothalamic-pituitary-adrenal axis (HPA). Cortisolsecretion has a circadian release rhythm with peak values in earlymorning and trough values at midnight.

The production and secretion of the most important glucocorticoid,cortisol, is governed by a complex and highly efficient system thatincludes the hypothalamus, pituitary and the adrenal glands i.e.,hypothalamic-pituitary-adrenal axis. Cortisol secretion is regulated bythe suprachiasmatic nucleus of the hypothalamus into a circadian releaserhythm. The timing is synchronized with the solar day by dark-lightshifts, which normally reflect the habitual sleep-wake pattern.Therefore in healthy persons, the cortisol secretion has a 24-hourcircadian pattern with peak serum levels in the early morning, 3-6 hoursafter onset of sleep, and nadir levels around midnight. Physical andpsychological stressors also activate cortisol secretion. Changedpatterns of serum cortisol levels have been observed in connection withabnormal adrenocorticotropic hormone (ACTH), levels, clinicaldepression, psychological stress, and physiological stressors such ashypoglycemia, illness, fever, trauma, surgery, fear, pain, physicalexertion, or temperature extremes. Cortisol levels and responsivenessmay also differ from normal for elderly individuals and in individualswith autism or Asperger's syndrome.

Glucocorticoids (GCs) such as, in humans, cortisol, perform severalimportant functions. These include participating in the regulation ofcarbohydrate, protein and fat metabolism by signaling the liver to makeglucose and glycogen, the adipose tissues to release lipids and fattyacids into the bloodstream, and the skeletal muscles to release proteinsor amino acids into the bloodstream. GCs also decrease bone formation.

GCs also regulate the body's inflammatory response as well. GCs are partof the feedback mechanism in the immune system that inhibits immuneactivity (i.e., inflammation). GCs cause their effects by binding to theGCR. The activated GCR complex in turn up-regulates the expression ofanti-inflammatory proteins in the nucleus (a process known astransactivation) and represses the expression of pro-inflammatoryproteins in the cytosol by preventing the translocation of othertranscription factors from the cytosol into the nucleus(transrepression) (Rhen T and Cidlowski J A. NEJM 2005; 353: 1711-23).

GCR antagonist therapy is helpful in patients with abnormally highlevels of cortisol (but maintained circadian rhythm), overresponsiveness to normal levels, or high night time cortisol levels as afeature of disrupted circadian rhythm. Such altered cortisol physiologymay relate to acute or chronic stress (e.g. related to physical orpsychological trauma) or as an age related change in elderlyindividuals. Successful therapeutic use of such agents is thus oftendependent on determining circadian cortisol levels (either peak levelsduring the day, e.g., at noon, or measurements taken every 4 hours or 6hours over a 24 hour period). This combined system of salivary cortisolquantification as an enabling device for its paired GCR antagonist willidentify individuals for whom GCR antagonist therapy has a benefit.

The glucocorticoid receptor (GR) is expressed at high levels in somenormal tissues, but not in others. Likewise, malignant tumors of diversetypes and sites have variable GR expression. When present in normal ortumor (benign or malignant) tissues, this GR expression may be variouslylocated in some or all of their cellular sub-compartments: 1. stemcells; 2. progenitor (so called “transit amplifying”) cell descendentsof activated stem cells; and 3. differentiated progeny of activated stemor progenitor cells.

The present invention therefore relates to the use of GR antagonists(e.g., ORG 34517—a relatively specific GR antagonist, RU486—anon-specific GR antagonist, and others), optionally in combination withat least one other agent, for treating or preventing treatment resistantprostate cancer, treating or preventing neoplasia, and/or treating orpreventing infection related to acute or chronic injury or disease.

All references cited herein are incorporated herein by reference intheir entireties.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method for treating neoplasia characterized byexpression of a glucocorticoid receptor, in a patient in need of suchtreatment, comprising: administering to said patient at least onetherapeutically effective neoplasia-treating therapy, and a GCR(glucocorticoid receptor) antagonist selected from the group consistingof ORG 34517, 11-(substituted phenyl)-estra-4,9-diene derivatives, and11-(substituted phenyl)-estra-4,9-diene derivatives of formula I

wherein A is a residue of a 5- or 6-membered ring containing 2heteroatoms which are not connected to each other and independentlyselected from O and S, the ring being optionally substituted with one ormore halogen atoms, or A is a residue of a 5- or 6-membered ring whereinno double C—C bonds are present, containing 1 heteroatom selected from Oand S, which heteroatom is connected to the phenyl group at the positionindicated with an asterisk, the ring being optionally substituted withone or more halogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H,(1-8C)alkyl, halogen or CF3; X is selected from (H₂OH), 0, and NOH; andthe interrupted line represents an optional bond, thereby treating saidanimal or human. The invention provides a method for treating neoplasiacharacterized by expression of a glucocorticoid receptor wherein the GCRantagonist is ORG34517(11β,17β-11-(1,3-benzodioxol-5-yl)-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one) which has the following formula:

The invention provides a method for treating neoplasia characterized byexpression of a glucocorticoid receptor wherein the neoplasia isselected from the group consisting of hepatocellular carcinoma,esophageal squamous cell carcinoma, breast cancer, pancreatic cancer,squamous cell cancer or adenocarcinoma of the head and neck, colorectalcancer, renal cancer, brain cancer, prostate cancer, small and non-smallcell lung cancer, bladder cancer, bone or joint cancer, uterine cancer,cervical cancer, multiple myeloma, hematopoietic malignancies, lymphoma,Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, melanoma,squamous cell carcinoma, leukemia, lung cancer, ovarian cancer, stomachcancer, Kaposi's sarcoma, laryngeal cancer, endocrine carcinomas, cancerof the thyroid gland, cancer of the parathyroid gland, cancer of thepituitary gland, cancer of the adrenal gland, and combinations thereof.The invention provides a method for treating neoplasia characterized byexpression of a glucocorticoid receptor wherein the neoplasia ischemo-resistant ER/GR+ breast cancer. The invention provides a methodfor treating neoplasia characterized by expression of a glucocorticoidreceptor wherein said neoplasia-treating therapy is radiation. Theinvention provides a method for treating neoplasia characterized byexpression of a glucocorticoid receptor wherein said neoplasia-treatingtherapy is a radionuclide. The invention provides a method for treatingneoplasia characterized by expression of a glucocorticoid receptorwherein said neoplasia treating therapy is selected from the groupconsisting of X-rays, gamma rays, and other sources of radiation. Theinvention provides a method for treating neoplasia characterized byexpression of a glucocorticoid receptor wherein said neoplasia treatingtherapy is radiation therapy administered as external beam radiation,wherein the radiation is directed from a remote source. The inventionprovides a method for treating neoplasia characterized by expression ofa glucocorticoid receptor wherein said neoplasia treating therapy isradiation therapy administered as brachytherapy wherein a radioactivesource is placed inside the body close to cancer stem cells, cancercells, and/or a tumor mass. The invention provides a method for treatingneoplasia characterized by expression of a glucocorticoid receptorwherein the method comprises administration of a neoplasia treatingtherapy followed by the administration of the GCR antagonist ORG34517.The invention provides a method for treating neoplasia characterized byexpression of a glucocorticoid receptor wherein the method comprisesadministration of a neoplasia treating therapy and the administration ofthe GCR antagonist ORG34517 concurrently. The invention provides amethod for treating neoplasia characterized by expression of aglucocorticoid receptor wherein the method comprises administration ofthe GCR antagonist ORG34517 followed by the administration of aneoplasia treating therapy. The invention provides a method for treatingneoplasia characterized by expression of a glucocorticoid receptorwherein said neoplasia treating therapy comprises both radiation therapyand an additional neoplasia treating therapy. The invention provides amethod for treating neoplasia characterized by expression of aglucocorticoid receptor wherein said additional neoplasia treatingtherapy is selected from the group consisting of peptides, polypeptides,fusion proteins, nucleic acid molecules, small molecules, mimeticagents, synthetic drugs, inorganic molecules, organic molecules,hormonal therapies, anti-angiogenesis therapies, targeted therapies,biological therapies, immunotherapy, and surgery. The invention providesa method for treating neoplasia characterized by expression of aglucocorticoid receptor wherein the administration of ORG 34517 forGR-blockade, reduces toxicities and side effects when givensystemically. The invention provides a method for treating neoplasiacharacterized by expression of a glucocorticoid receptor wherein the ORG34517 given systemically through oral or intravenous routes. Theinvention provides a method for treating neoplasia characterized byexpression of a glucocorticoid receptor wherein the ORG 34517 is givento accomplish cure or remission of tumor. The invention provides amethod for treating neoplasia characterized by expression of aglucocorticoid receptor wherein the ORG 34517 is given to accomplishreduction of tumor burden to enhance effectiveness of subsequentsurgical resection.

The invention provides a method for treating neoplasia in a patient inneed of such treatment, the method comprising: a) administering to saidpatient a therapeutically effective amount of ORG 34517; and b)administering to said patient at least one therapeutically effectiveneoplasia-treating therapy, thereby treating said patient. The inventionprovides a method for treating neoplasia wherein the neoplasia isselected from the group consisting of hepatocellular carcinoma,esophageal squamous cell carcinoma, breast cancer, pancreatic cancer,squamous cell cancer or adenocarcinoma of the head and neck, colorectalcancer, renal cancer, brain cancer, prostate cancer, small and non-smallcell lung cancer, bladder cancer, bone or joint cancer, uterine cancer,cervical cancer, multiple myeloma, hematopoietic malignancies, lymphoma,Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, melanoma,squamous cell carcinoma, leukemia, lung cancer, ovarian cancer, stomachcancer, Kaposi's sarcoma, laryngeal cancer, endocrine carcinomas, cancerof the thyroid gland, cancer of the parathyroid gland, cancer of thepituitary gland, cancer of the adrenal gland, and combinations thereof.The invention provides a method for treating neoplasia wherein theneoplasia is chemo-resistant ER/GR+ breast cancer. The inventionprovides a method for treating neoplasia wherein said neoplasia treatingtherapy is radiation. The invention provides a method for treatingneoplasia wherein said neoplasia treating therapy is a radionuclide. Theinvention provides a method for treating neoplasia wherein saidneoplasia treating therapy is selected from the group consisting ofX-rays, gamma rays, and other sources of radiation. The inventionprovides a method for treating neoplasia wherein said neoplasia treatingtherapy is radiation therapy administered as external beam radiation orteletherapy, wherein the radiation is directed from a remote source. Theinvention provides a method for treating neoplasia wherein saidneoplasia treating therapy is radiation therapy administered as internaltherapy or brachytherapy wherein a radioactive source is placed insidethe body close to cancer stem cells, cancer cells, and/or a tumor mass.The invention provides a method for treating neoplasia wherein themethod comprises administration of a neoplasia treating therapy followedby the administration of the GCR antagonist ORG34517. The inventionprovides a method for treating neoplasia wherein the method comprisesadministration of a neoplasia treating therapy and the administration ofthe GCR antagonist ORG34517 concurrently. The invention provides amethod for treating neoplasia wherein the method comprisesadministration of the GCR antagonist ORG34517 followed by theadministration of the neoplasia treating therapy. The invention providesa method for treating neoplasia wherein said neoplasia treating therapycomprises both radiation therapy and an additional neoplasia treatingtherapy. The invention provides a method for treating neoplasia whereinsaid additional neoplasia treating therapy is selected from the groupconsisting of peptides, polypeptides, fusion proteins, nucleic acidmolecules, small molecules, mimetic agents, synthetic drugs, inorganicmolecules, organic molecules, hormonal therapies, anti-angiogenesistherapies, targeted therapies, biological therapies, immunotherapy, andsurgery. The invention provides a method for treating neoplasia whereinthe neoplasia expresses multidrug resistance genes when GR is activatedthrough binding by endogenous cortisol. The invention provides a methodfor treating neoplasia wherein the ORG 34517 given systemically throughoral or intravenous routes. The invention provides a method for treatingneoplasia wherein the ORG 34517 is given to accomplish cure or remissionof tumor. The invention provides a method for treating neoplasia whereinthe ORG 34517 is given to accomplish reduction of tumor burden toenhance effectiveness of subsequent surgical resection.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 is an example of a sample carrier.

FIG. 2 is an example of glass reaction vessel with reagent one dispensedinside.

FIG. 3 is an example of a fluorescent ligand reagent two, dryformulation to be dissolved in the reaction vessel.

FIGS. 4A through 4D show examples of additional embodiments of thechemistry process configuration. FIG. 4A shows a blister pack of reagentcartridge units. FIG. 4B shows an exemplary reaction vessel and cap.FIG. 4C shows an exemplary sample carrier. FIG. 4D shows an exemplaryreagent 2 (fluorescent ligand) carrier.

FIGS. 5A through 5C show examples of additional embodiments of thechemistry process configuration. FIG. 5A shows an exemplary samplecarrier. FIG. 5B shows an exemplary reaction vessel and cap. FIG. 5Cshows an exemplary reagent 2 (fluorescent ligand) carrier.

FIGS. 6A and 6B show additional embodiments of chemistry processconfiguration-plastic cartridge. FIG. 6A shows an exemplary samplecarrier. FIG. 6B shows an exemplary reagent 2 (fluorescent ligand)carrier.

FIG. 7 is an example of a Fluorescence Polarization Reader (DC andbattery operated wt.<3 lbs).

FIG. 8 is Effect of Vehicle (Ethanol/Castor Oil)/Vehicle (Ethanol/SesameOil), Vehicle (Ethanol/Castor Oil)/Taxol (10 mg/kg/day) and ORG 34517(20.5 mg/kg/day)/Taxol (10 mg/kg/day) on relative tumor growth.Compounds are administered day 1-5.

FIG. 9 shows the Effect of Vehicle (Ethanol/Castor Oil)/Taxol (10mg/kg/day) and ORG 34517 (20.5 mg/kg/day)/Taxol (10 mg/kg/day) onrelative tumor growth. The average curve for Veh/Taxo is shown.517/Taxocurves are show for each individual animal. Compounds areadministered day 1-5.

FIG. 10 shows the results of an experiment in which mice were implantedwith cultured ER-GR+ human breast cancer cells. As tumor volume in eachmouse approached the test threshold of 200 mm3, the mice were randomizedto receive intraperitoneal injections of vehicle alone, chemotherapy(Paclitaxel) alone, and chemotherapy and ORG 34517. Each group contained3 mice. Results show significant differences in attained tumor volume

FIG. 11 shows the AUC₀₋₂₄. Two dogs were included in the test. Thenanosuspension increases the exposure of(11b,17b)-11-(1,3-benzodioxol-5-yl)-17-hydroxy-17-(1-propynyl)estra-4,9-d-ien-3-one.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to the use of, for example, a glucocorticoidreceptor antagonist, optionally in combination with another agent, fortreating or preventing treatment resistant prostate cancer, treating orpreventing neoplasia, and/or treating or preventing infection related toacute or chronic injury or disease.

As used herein, the term “effective amount” refers to the amount of atherapy that is sufficient to result in the prevention of thedevelopment, recurrence, or onset of a disease or condition, such asneoplasia or infection, and one or more symptoms thereof, to enhance orimprove the prophylactic effect(s) of another therapy, reduce theseverity, the duration of a disease or condition, such as neoplasia orinfection, ameliorate one or more symptoms of a disease or conditionsuch as neoplasia or infection, prevent the advancement of a disease orcondition, such as neoplasia or infection, cause regression of a diseaseor condition, such as neoplasia or infection, and/or enhance or improvethe therapeutic effect(s) of another therapy.

As used herein, the phrase “pharmaceutically acceptable” means approvedby a regulatory agency of the federal or a state government, or listedin the U.S. Pharmacopeia, European Pharmacopeia, or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans.

As used herein, the terms “prevent,” “preventing” and “prevention” inthe context of the administration of a therapy to a subject refer to theprevention or inhibition of the recurrence, onset, and/or development ofa disease or condition, such as neoplasia or infection, or a symptomthereof in a subject resulting from the administration of a therapy(e.g., a prophylactic or therapeutic agent), or a combination oftherapies (e.g., a combination of prophylactic or therapeutic agents).

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the term “patient” refers to an animal,preferably a mammal such as a non-primate (e.g., cows, pigs, horses,cats, dogs, rats etc.) and a primate (e.g., monkey and human), and mostpreferably a human. In some embodiments, the subject is a non-humananimal such as a farm animal (e.g., a horse, pig, or cow) or a pet(e.g., a dog or cat). In a specific embodiment, the subject is anelderly human. In another embodiment, the subject is a human adult. Inanother embodiment, the subject is a human child. In yet anotherembodiment, the subject is a human infant.

As used herein, the terms “therapies” and “therapy” can refer to anymethod(s), composition(s), and/or agent(s) that can be used in theprevention, treatment and/or management of a disease or condition, suchas neoplasia or infection, or one or more symptoms thereof.

As used herein, the terms “treat,” “treatment,” and “treating” in thecontext of the administration of a therapy to a subject refer to thereduction or inhibition of the progression and/or duration of a diseaseor condition, such as neoplasia or infection, the reduction oramelioration of the severity of a disease or condition, such asneoplasia or infection, and/or the amelioration of one or more symptomsthereof resulting from the administration of one or more therapies.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

The term “androgenergic antagonist” refers to agents that can preventandrogens from expressing their biological effects on responsivetissues. These agents alter the androgen pathway by blocking theappropriate receptors, competing for binding sites on the cell'ssurface, or affecting androgen production. Androgenergic antagonist canbe prescribed to treat an array of diseases and disorders. In men, theseagents are most frequently used to treat prostate cancer. In women,these agents are used to decrease levels of male hormones causingsymptoms of hyperandrogenism. Androgenergic antagonist present in theenvironment have become a topic of concern. Many industrial chemicals,pesticides and insecticides exhibit antiandrogenic effects. Non-limitingexamples of the androgenergic antagonist include, but not limited to,allylestrenol, oxendolone, osaterone acetate, bicalutamide, steroidal,anti-androgergic agents, medroxyprogesterone (MPA), cyproterone,cyproterone acetate (CPA), dienogest, flutamide, nilutamide,spironolactone, 5alpha-reductase inhibitors, dutasteride, finasteride,salts thereof, gold nanoparticles thereof, combinations thereof, and thelike. In some embodiments of the present invention, examples of theandrogenergic antagonist includes, but not limited to a goldnanoparticle of alpha-bicalutamide, or a gold nanoparticle ofbeta-bicalutamide.

Glucocorticoid Receptor

The glucocorticoid receptor is widely distributed and expressed in manycultured cell lines, and the control of gene expression byglucocorticoids, therefore, has been widely studied as a model fortranscriptional regulation. A number of glucocorticoid-responsivetranscription units, including mouse mammary tumor virus (MMTV)(Ringold, et al., 1975; Parks, et al., 1974), mouse and humanmetallothionein (Eager, et al., 1981; Karin, et al., 1980), ratalpha₂M-globulin (Kurtz, et al., 1977) and rat and human growth hormone(Spindler, et al., 1982; Evans, et al., 1982; Robins, et al., 1982)genes have been identified. DNA sequences mediating transcriptionalstimulation of several of these genes have been localized. For MMTV,these sequences are discrete genomic regions upstream of thetranscriptional start site which appear to exert their actionsindependently of orientation and position (Chandler, et al., 1983;Ostrowski, et al., 1984). The steroid/receptor complex appears to bindto these regulatory sequences and purified receptor has been used todefine the specific binding sites (Govinda, et al., 1982; Scheidereit,et al., 1983; Pfahl, 1982; Payvar, et al., 1983). The ability of theglucocorticoid-responsive element (GRE) to alter its position andorientation yet still maintain promoter inducibility suggests that itresembles the class of cis-acting regulatory sequences termed enhancers(Chandler, et al., 1983). First discovered in viruses and subsequentlyin cellular genes, these sequences are necessary for efficienttranscription in vivo (Laimonis, et al., 1982; Benoist, et al., 1981;Baerji, et al., 1983). It has been suggested that enhancers arerecognized by trans-acting factors that mediate regulatory effects bytissue-specific transcriptional control. Although the enhancer factorshave not been well characterized, the glucocorticoid receptor may serveas a paradigm for these putative gene activator proteins.

It is generally accepted that the unliganded glucocorticoid receptor(GR) resides in the cytoplasm, and that hormone activation leads both tonuclear accumulation and gene activation. (Gasc, J. -M. & Baulieu, E. E.(1987) in Steroid Hormone Receptors: Their Intracellular Localisation,ed. Clark, C. R. (Ellis Horwood Ltd., Chichester, England), pp. 233-250;Beato, M. (1989) Cell 56, 335-344; Carson-Jurica, M. A., Schrader, W. T.& O'Malley, B. W. (1990) Endocr. Rev. 11, 201-220; Gronemeyer, H. (1993)in Steroid Hormone Action, ed. Parker, M. G. (Oxford University Press,New York), pp. 94-117; Tsai, M. J. & O'Malley, B. W. (1994) Annu. Rev.Biochem. 63, 451-486; Akner, G., Wikstrom, A. C. & Gustafsson, J. A.(1995) J. Steroid Biochem. Mol. Biol. 52, 1-16), and references therein.However, the mechanisms involved in nuclear translocation and targetingof steroid receptors to regulatory sites in chromatin have been poorlyunderstood. It has previously been difficult to discriminate between theability of a given receptor mutant, or a given receptor/ligandcombination, to participate in the separate processes of receptoractivation, nuclear translocation, sequence-specific binding, andpromoter activation.

The glucocorticoid receptor (GR) is expressed in a subset of bothERalpha-positive and -negative human breast cancers as well as in someovarian cancers. In vitro and in vivo experiments suggest thatactivation of the GR in ER-negative pre-malignant breast epithelial andcancer cells initiates cell survival pathways under stress conditionsthat normally induce significant cell death (e.g. chemotherapy,radiation, growth factor deprivation).

The glucocorticoid receptor (GR) is present in glucocorticoid responsivecells where it resides in the cytosol in an inactive state until it isstimulated by an agonist. Upon stimulation the glucocorticoid receptortranslocates to the cell nucleus where it specifically interacts withDNA and/or protein(s) and regulates transcription in a glucocorticoidresponsive manner. Two examples of proteins that interact with theglucocorticoid receptor are the transcription factors, API and NFκ-B.Such interactions result in inhibition of API- and NFκ-B-mediatedtranscription and are believed to be responsible for some of theanti-inflammatory activity of endogenously administered glucocorticoids.In addition, glucocorticoids may also exert physiologic effectsindependent of nuclear transcription. Biologically relevantglucocorticoid receptor agonists include cortisol and corticosterone.Many synthetic glucocorticoid receptor agonists exist includingdexamethasone, prednisone and prednisolone.

Glucocorticoid Receptor Antagonists

The composition and methods of the invention may make use ofglucocorticoid receptor antagonists. Glucocorticoid receptor antagonistsbind to the receptor and prevent glucocorticoid receptor agonists frombinding and eliciting GR mediated events, including transcription. RU486is an example of a non-selective glucocorticoid receptor antagonist.

Compounds having high glucocorticoid receptor binding affinity and, inaddition, high in vivo anti-glucocorticoid activity, while having, forexample, low androgenic and progestagenic activities are disclosed inU.S. Pat. No. 6,011,025, incorporated herein by reference in itsentirety. ORG 34517 is an example of a compound with high glucocorticoidreceptor binding affinity while having low androgenic and progestagenicactivities.

It has been found that 11-(substituted phenyl)-estra-4,9-dienederivatives of formula I

whereinA is a residue of a 5- or 6-membered ring containing 2 heteroatoms whichare not connected to each other and independently selected from O and S,the ring being optionally substituted with one or more halogen atoms, orA is a residue of a 5- or 6-membered ring wherein no double C—C bondsare present, containing 1 heteroatom selected from O and S, whichheteroatom is connected to the phenyl group at the position indicatedwith an asterisk, the ring being optionally substituted with one or morehalogen atoms; R1 is H or 1-oxo(1-4C)alkyl; R2 is H, (1-8C)alkyl,halogen or CF3; X is selected from (H₂OH), O, and NOH; and theinterrupted line represents an optional bond, show specific and highglucocorticoid receptor binding affinity and are highly active in vivoshowing predominant anti-glucocorticoid activity.

The compounds lack appreciable affinity for mineralocorticoid,progesterone, estrogen and androgen receptors, indicating a clean sideeffect profile.

The 11-(substituted phenyl)-estra-4,9-diene derivatives of the inventioncan be used in the prevention and treatment of glucocorticoid dependentdiseases or symptoms, like Cushing syndrome, diabetes, glaucoma, sleepdisturbances, depression, anxiety, atherosclerosis, hypertension,adiposity, osteoporosis and withdrawal symptoms from narcotics and theirmixtures.

Preferred compounds according to this invention are 11-(substitutedphenyl) estra-4,9-diene derivatives, wherein the heteroatom(s) are (is)O, the 5- or 6-membered ring being optionally substituted with one ormore fluorine atoms; R1 is H; and X is O or NOH. More preferredcompounds are 11-(substituted phenyl) estra-4,9-diene derivativeswherein A is a residue of a 5-membered ring. Particularly preferred are11-(substituted phenyl) estra-4,9-diene derivatives wherein A contains 2heteroatoms being O.

Especially preferred are 11-(substituted phenyl) estra-4,9-dienederivatives wherein R2 is methyl and the interrupted line represents abond.

The most preferred compound is(11β,17β)-11-(1,3-benzodioxol-5-yl)-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one (ORG 34517).

The term halogen means a fluorine, chlorine, bromine or iodine atom.Fluorine is the preferred halogen in ring A and when R2 is halogen,chlorine is preferred.

The terms (1-4C)alkyl and (1-8C)alkyl, as used in the definitions of R1and R2, respectively, mean alkyl groups having 1-4 and 1-8 carbon atoms,respectively, for example methyl, ethyl, propyl, isopropyl, butyl,sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, octyl.

The 11-(substituted phenyl)-estra-4,9-diene derivatives according to thepresent invention can be prepared by a process wherein a compound offormula II

wherein A, R2 and the interrupted line have the meanings as previouslydefined, R1 is H, and P is a protected keto-group, is dehydrated anddeprotected, after which the 17β-OH is optionally esterified by reactionwith an appropriate carboxylic acid to give a derivative wherein R1 is1-oxo(1-4C)alkyl, and optionally the 3-oxo group is converted into thecorresponding 3-hydroxy- or 3-oxime derivative. The 3-oxo group can bereduced to form the 3-hydroxy-derivative by using a suitable reducingagent, such as sodium borohydride. The 3-oxime derivatives can beprepared by hydroxylamine treatment in a suitable solvent, likepyridine.

The derivatives of formula II may be prepared according to well knownmethods described and used for the preparation of steroids.

A suitable process for the preparation of derivatives of formula IIstarts from estra-4,9-diene-3,17-dione. Selective reduction of the17-keto group to 17ß-OH, 17α-H, e.g. with sodium borohydride, followedby protection of the 3-keto group, e.g., by ketalisation withethyleneglycol, triethylorthoformate and p-toluenesulfonic acid, andoxidation of the 17-hydroxy group, e.g. with pyridinium chlorochromate,provides the 3-ketoprotected estra-5(10),9(11)-diene-3,17-dione.Alkynylation at the 17-position (yielding a 17α-alkynyl,17ß-OHderivative), followed by epoxidation of the 5(10) double bond, e.g. withhydrogen peroxide, trifluoroacetophenone, and pyridine indichloromethane according to the method as disclosed in European patentapplication EP 0 298 020, provides the 3-ketoprotected5α,10α-epoxy-|7α-alkynyl-17 ß-hydroxy-estr-9(11)-ene-3-one.

Subsequently, compounds of formula II are formed from this epoxidederivative, for example by reaction with an organometallic compound ofthe formula

wherein X is a (alkali)metal, like lithium, or a magnesiumhalide,preferably magnesium bromide.

Suitable protective groups and methods to remove these groups are knownin the art, for example from T.W. Green: Protective Groups in OrganicSynthesis (Wiley, N Y, 1981). Particularly suitable protective groupsfor the protection of keto groups are acetals, e.g. 1,2-ethylene ketal.

The specificity of ORG 34517 for GR blockade, without significantcross-binding to other related steroidal hormone receptors (such asthose for estrogen and progesterone), eliminates the likelihood ofsignificant toxicities and side effects. Indeed, none were identified inall the substantial phase I and phase II clinical trials that alreadyhave been performed with the compound. Because the drug is envisioned asbeing used in limited dosing over time, coordinated with theintermittent dosing strategies typical for chemotherapeutic agents, theGR blockade also would not lead to significant alteration of HPA-axisfunctioning, with rapid restitution of the HPA-axis to baselinefollowing dosing.

Androgen Receptor Antagonists

The compositions and methods of the invention may also make use of oneor more androgen receptor antagonists, such as in a combination with theglucocorticoid receptor antagonist of the invention. For example, theinvention provides with at least one glucocorticoid receptor antagonistin combination with at least one androgen receptor antagonist, such asfor example, ARN 509(4-{7-[6-Cyano-5-(trifluoromethyl)-3-pyridinyl]-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl}-2-fluoro-N-methylbenzamide).ARN-509 is a novel androgen receptor (AR) antagonist for the treatmentof castration-resistant prostate cancer (CRPC). ARN-509 inhibits ARnuclear translocation and AR binding to androgen response elements and,unlike bicalutamide, does not exhibit agonist properties in the contextof AR overexpression.

Another exemplary antiadrogen is bicalutamide, which has the chemicalname(R,S)—N-(4-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3-(triflu-oromethyl)propanamide,Flutamide (brand name Eulexin), nilutamide (brand names Anandron andNilandron) and bicalutamide (brand name Casodex) are nonsteroidal,“pure” antiandrogens; 5-alpha-reductase inhibitors such as finasteride(brand names Proscar and Propecia), dutasteride (brand name Avodart),bexlosteride, izonsteride, turosteride, and epristeride areantiandrogenic as they prevent the conversion of testosterone todihydrotestosterone (DHT); Spironolactone (brand names Aldactone andSpirotone), a synthetic 17-spirolactone corticosteroid; Cyproteroneacetate (brand names Androcur, Climen, Diane 35, and Ginette 35) is asynthetic steroid, a potent antiandrogen that also possessesprogestational properties. Hydroxyflutamide.

In some embodiments, steroidal or nonsteroidal androgen receptorantagonists include but are not limited to flutamide, hydroxyflutamide,bicalutamide, nilutamide, or hydroxysteroid dehydrogenase inhibitor.

In one embodiment, the androgen receptor antagonist is enzalutamide(marketed as Xtandi®, Astellas Pharma US, Inc.), also known as andreferred to herein as MDV3100, having the chemical name4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimida-zolidin-1-yl)-2-fluoro-N-methylbenzamide.

The compositions and methods of the invention may also make use of oneor more androgen receptor antagonist, such as in a combination with theglucocorticoid receptor antagonist of the invention. The androgenreceptor antagonist may be selected from the group consisting of, forexample, flutamide, nilutamide, enzalutamide, bicalutamide, ketonazole,abiraterone, abiraterone acetate, orteronel, finasteride, dutasteride,bexlosteride, izonsteride, turosteride, episteride, dexamethasone,prednisone, leuprolide, goserelin, triptorelin, histrelin, estrogen,MDV3100, Cyproterone acetate, Spironolactone, flutamide,hydroxyflutamide, and combiations thereof.

The selective androgen receptor (AR) antagonists embodied herein haveutility for numerous conditions and diseases such as but not limited tomale contraception; treatment of a variety of male hormone-relatedconditions such as hypersexuality and sexual deviation; treatment ofconditions including benign prostatic hyperplasia, acne vugaris,androgenetic alopecia, and hirsutism; purposefully preventing orcounteracting masculinisation in the case of transsexual womenundergoing sex reassignment therapy; an antineoplastic agent andpalliative, adjuvant or neoadjuvant hormonal therapy in prostate cancer;and decreasing the incidence of, halting or causing a regression ofprostate cancer.

Prostate cancer is one of the most common cancers in men around theworld, and is one of the leading causes of cancer death in men in theUnited States. The androgen receptor antagonist drugs, such as flutamideand bicalutamide, were originally designed to avoid the side effects ofHT but androgen agonism was observed for hydroxyfluamide (the activeform of flutamide) and bicalutamide. The compositions of the presentinvention are combinations of GCRAs and, for example, androgen receptorantagonists, which can be used to alleviate any condition associatedwith inappropriate activation of the androgen receptor. In addition toprostate cancer, other examples of such conditions include acne,hirsutism, seborrhea, excess sebum, and alopecia. In order to exhibitthe therapeutic properties described above, the compounds need to beadministered in a quantity sufficient to inhibit activation of theandrogen receptor. In a typical embodiment, the compounds areadministered topically, which is especially appropriate for hirsutism,alopecia, acne and hyperseborhhea. Androgens, having a profound effecton hair loss, stimulate hair growth by prolonging the growth phase ofthe hair cycle (anagen) and increasing follicle size. Hair growth on thescalp does not require androgens but, paradoxically, androgens arenecessary for balding on the scalp in genetically predisposedindividuals (androgenic alopecia) where there is a progressive declinein the duration of anagen and in hair follicle size. The compositions ofthe invention may also be used topically to decrease seborrheaproduction and more specifically to alleviate hyperseborrhoea (oilyskin), which can be used topically alleviate acne.

Poly(ADP-Ribose) Polymerase (PARP)

The compositions and methods of the invention may also make use of oneor more PARP inhibitors, such as in a combination with theglucocorticoid receptor antagonist of the invention. The poly(ADP-ribose) polymerase (PARP) is also known as poly (ADP-ribose)synthase and poly ADP-ribosyltransferase. PARP catalyzes the formationof mono- and poly (ADP-ribose) polymers which can attach to cellularproteins (as well as to itself) and thereby modify the activities ofthose proteins. The enzyme plays a role in regulation of transcription,cell proliferation, and chromatin remodeling (see D'amours et al.,Biochem., 342: 249268, 1999).

Poly(ADP-ribose)polymerase has an essential role in facilitating DNArepair, controlling RNA transcription, mediating cell death, andregulating immune response. These actions make PARP inhibitors targetsfor a broad spectrum of disorders. (Virag L., et al., Pharmacol. Rev.2002 54(3):375-429). In various preclinical cancer models and humanclinical trials, PARP inhibitors have been shown to potentiate radiationand chemotherapy by increasing apoptosis of cancer cells, limiting tumorgrowth, decreasing metastasis, and prolonging the survival oftumor-bearing subjects. (WO 2007-084532; Donawho C. K., et al., ClinCancer Res 2007 13(9):2728-37; Kummar S., et al., J Clin Oncol. 200927(16):2705-11).

PARP comprises an N-terminal DNA binding domain, an automodificationdomain, and a C-terminal catalytic domain. Various cellular proteinsinteract with PARP. The N-terminal DNA binding domain contains two zincfinger motifs. Transcription enhancer factor-1 (TEF-1), retinoid Xreceptor a, DNA polymerase a, X-ray repair cross-complementing factor-1(XRCC1) and PARP itself interact with PARP in this domain. Theautomodification domain contains a BRCT motif, one of theprotein-protein interaction modules. This motif is originally found inthe C-terminus of BRCA1 (breast cancer susceptibility protein 1) and ispresent in various proteins related to DNA repair, recombination andcell-cycle checkpoint control. POU-homeodomain-containing octamertranscription factor-1 (Oct-1), YinYang (YY)1, and ubiquitinconjugatingenzyme 9 (ubc9) could interact with this BRCT motif in PARP.

More than 15 members of the PARP family of genes are present in themammalian genome. PARP family proteins and poly(ADP-ribose)glycohydrolase (PARG), which degrades poly(ADP-ribose) to ADP-ribose,are involved in a variety of cell regulatory functions including DNAdamage response and transcriptional regulation and associated withcarcinogenesis and the biology of cancer.

Several PARP family proteins have been identified. Tankyrase has beenfound as an interacting protein of telomere regulatory factor 1 (TRF-1)and is involved in telomere regulation. Vault PARP (VPARP) is acomponent in the vault complex, which acts as a nuclear-cytoplasmictransporter. PARP-2, PARP-3 and 2,3,7,8-tetrachlorodibenzo-p-dioxininducible PARP (TiPARP) have also been identified. Therefore,poly(ADP-ribose) metabolism could be related to a variety of cellregulatory functions.

A member of this gene family is PARP-1. The PARP-1 gene product isexpressed at high levels in the nuclei of cells and is dependent uponDNA damage for activation. It is believed that PARP-1 binds to DNAsingle or double-stranded breaks (DSBs) through an amino-terminalDNA-binding domain. The binding activates the carboxy-terminal catalyticdomain and results in the formation of polymers of ADP-ribose on targetmolecules. PARP-1 is itself a target of poly ADP-ribosylation by virtueof a centrally located automodification domain. The ribosylation ofPARP-1 causes dissociation of the PARP-1 molecules from the DNA. Theentire process of binding, ribosylation, and dissociation occurs veryrapidly. It has been suggested that this transient binding of PARP-1 tosites of DNA damage results in the recruitment of DNA repair machineryor may act to suppress the recombination long enough for the recruitmentof repair machinery.

The source of ADP-ribose for the PARP reaction is nicotinamide adenosinedinucleotide (NAD). NAD is synthesized in cells from cellular ATP storesand thus high levels of activation of PARP activity can rapidly lead todepletion of cellular energy stores. It has been demonstrated thatinduction of PARP activity can lead to cell death that is correlatedwith depletion of cellular NAD and ATP pools. PARP activity is inducedin many instances of oxidative stress or during inflammation. Forexample, during reperfusion of ischemic tissues reactive nitric oxide isgenerated and nitric oxide results in the generation of additionalreactive oxygen species including hydrogen peroxide, peroxynitrate, andhydroxyl radical. These latter species can directly damage DNA and theresulting damage induces activation of PARP activity. Frequently, itappears that sufficient activation of PARP activity occurs such that thecellular energy stores are depleted and the cell dies. A similarmechanism is believed to operate during inflammation when endothelialcells and pro-inflammatory cells synthesize nitric oxide, which resultsin oxidative DNA damage in surrounding cells and the subsequentactivation of PARP activity. The cell death that results from PARPactivation is believed to be a major contributing factor in the extentof tissue damage that results from ischemia-reperfusion injury or frominflammation.

PARP (poly-ADP ribose polymerase) participates in a variety ofDNA-related functions including cell proliferation, differentiation,apoptosis, DNA repair and also effects on telomere length and chromosomestability (d'Adda di Fagagna et al., Nature Gen., 23(1): 76-80, 1999).Oxidative stress-induced overactivation of PARP consumes NAD+ andconsequently ATP, culminating in cell dysfunction or necrosis. Thiscellular suicide mechanism has been implicated in the pathomechanism ofcancer, stroke, myocardial ischemia, diabetes, diabetes-associatedcardiovascular dysfunction, shock, traumatic central nervous systeminjury, arthritis, colitis, allergic encephalomyelitis, and variousother forms of inflammation. PARP has also been shown to associate withand regulate the function of several transcription factors.

PARP Inhibitors

Suitable PARP inhibitors for use in the compositions and methods of theinvention include, but are not limited to,4-[[3-[4-(cyclopropanecarbonyl)piperazine-1-carbonyl]-4-fluorophenyl]-met-hyl]-2H-phthalazin-1-one(Compound B, i.e., Olaparib), 4-iodo-3-nitrobenzamide (Iniparib),2-[(2R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide(ABT-888),8-Fluoro-2-{4-[(methylamino)methyl]-phenyl}-1,3,4,5-tetrahydro-6H-azepino-[5,4,3-cd]indol-6-one(AG014699), 4-methoxy-carbazole (CEP 9722),2-[4-[(3S)-piperidin-3-yl]phenyl]indazole-7-carboxamide hydrochloride(MK 4827), and 3-aminobenzamide, or a pharmaceutically acceptable saltthereof.

In some embodiments, the Glucocorticoid receptor antagonists, e.g., anORG-34517 composition described herein, is administered in combinationwith a poly ADP-ribose polymerase (PARP) inhibitor (e.g., BSI 201,Olaparib (AZD-2281), ABT-888, AG014699, CEP 9722, MK 4827, KU-0059436(AZD2281), LT-673, 3-aminobenzamide). Other example PARP inhibitorsinclude, i.e., pharmacological inhibitors of the enzyme poly ADP ribosepolymerase (PARP). Suitable PARP inhibitors may be iniparib, olaparib,rucaparib, veliparib, or CEP 9722.

Current PARP inhibitors in clinical trials include: Iniparib (Sanofi),Olaparib (AstraZeneca), Rucaparib (Pfizer), Veliparib (Abbott), CEP-9722(Cephalon), MK4827 (Merck), BMN-673 (Biomarin), among others.

Treatment Resistant Prostate Cancer

The invention provides a method for treating and/or preventing treatmentresistant prostate cancer in a patient in need of such treatment and/orprevention, comprising: administering to the patient therapeuticallyeffective amounts of a GCR (glucocorticoid receptor) antagonist asexemplified herein, in combination with, for example, a neoplasiatreating agent, including, for example, an androgen receptor (AR)antagonist. Prostate cancer is the most commonly diagnosed cancer in menand the second leading cause of death from cancer in North American andEuropean males. New therapeutic approaches are needed to prevent andtreat advanced and metastatic prostate cancer. Nutritional factors,particularly high intake of protein and calcium, as well as metabolicsyndrome, are known to modify prostate cancer risk and progression, butthe molecular mechanisms linking nutrition to prostate cancer areunknown. There are also links between prostate cancer and bonemetabolism. Osteocalcin (OC), which encodes a vitamin-K dependenthormone predominantly produced by osteoblasts/osteocytes in bone, whichfunctions to regulate energy metabolism, is also ectopically expressedby some prostate cancers that have a propensity to metastasize to bone.Polymorphisms in OC are also associated with prostate cancerprogression. Recent evidence has also identified a correlation betweenthe bone transcription factor Runx2 and advanced stages of prostate andbreast cancer, as evidenced by the effects of depletion of Runx2 by RNAinterference to inhibit migration and invasive properties of the cellsand prevent metastatic bone disease. It is possible that OC secreted bybone may directly target prostate cancer cells. Finally, androgendeprivation therapy is the principal medical therapy for prostatecancer, but androgen ablation often becomes ineffective in controllingprostate cancer progression and castration-resistant metastatic disease,particularly to bone, becomes incurable. There is growing evidence forthe presence of a putative membrane androgen sensing receptor thatmediates the rapid, non-genomic effects of androgens, which also mightbe involved in prostate cancer growth and metastasis. Regardless, cluesto possible new molecular targets to regulate prostate cancer growth andprogression may be discovered from a better understanding of themolecular mechanisms underlying nutritional risk factors, OC effects andandrogen resistance in prostate cancer.

Neoplasia, Cancer, Tumors, Proliferative Diseases, Malignancies andtheir Metastases

The invention provides a method for treating neoplasia in a patient inneed of such treatment, comprising: administering to the patienttherapeutically effective amounts of a GCR (glucocorticoid receptor)antagonist as exemplified herein, in combination with, for example, aneoplasia treating agent. The term “neoplasia” as used herein refersalso to tumors, proliferative diseases, malignancies and theirmetastases. Examples for cancer diseases are Adenocarcinomas of the headand neck (including salivary glands and oral cavity), gastrointestinaltract (including pharynx, esophagus, stomach, small intestine, largeintestine, anus), lung, liver (including hepatocellular carcinoma,cholangiocarcinoma, and mixed tumors), extrahepatic biliary tract andgallbladder, pancreas (including ductal and acinar types), genitourinarytracts (ovaries, fallopian tubes, endometrium, cervix, and vagina,ureters, urinary bladder, testicles, epididymis, prostate), and skinadnexa; squamous cell carcinomas of the head and neck (includingsalivary glands and oral cavity), gastrointestinal tract (includingpharynx, esophagus, anus), lung, intrahepatic and extrahepatic biliarytree (including gallbladder), pancreas, genitourinary tracts (includingendometrium, cervix, vagina, ureters, urinary bladder, testicles,epididymis, prostate), and skin adnexa; germ cell tumors (includingmalignant teratoma, embryonal carcinoma, struma ovarii, yolk sac tumor,seminoma, choriocarcinoma); sarcomas (including leiomyosarcomas,rhabdomyosarcomas, angiosarcomas, hemangioendotheliomas, liposarcomas,chondosarcomas, fibrosarcomas, Ewing sarcoma, malignant nerve sheathetumors, alveolar soft part sarcomas, clear cell sarcomas, synovialsarcoma, osteosarcomas); malignancies of the central nervous system(including astrocytomas, oligodendroglioma, glioblastoma,medulloblastoma); salivary gland malignancies (including adenoid cysticcarcinoma, adenosquamous carcinoma, clear cell carcinoma,cystadenocarcinoma, mucoepidermoid carcinoma); mixed type carcinomas(including hepatocellular-cholangiocarcinomas, carcinosarcomas, mixedadenoneurondocrine carcinomas, adenosquamous carcinomas); hepatocellularcarcinoma; blastic malignancies (including hepatoblastoma,neuroblastoma, ganglioneuroblastoma, nephroblastoma); renal cellcarcinomas; neuroendocrine carcinomas; thyroid carcinomas (includingpapillary, follicular, medullary, anaplastic carcinomas); parathyroidcarcinomas, pituitary gland carcinomas, adrenal gland carcinomas(including adrenocortical carcinomas, pheochromocytoma), andcombinations thereof.

Treating or Preventing Infection Related To Acute Or Chronic Injury OrDisease

The invention provides a method for treating or preventing infectionrelated to acute or chronic injury or disease in a patient in need ofsuch treatment, comprising: administering to the patient therapeuticallyeffective amounts of a GCR (glucocorticoid receptor) antagonist asexemplified herein, in combination with, for example, another agent. Inparticular, the invention has application to minimize life-threateningcomplications of persons suffering injury to cells, tissues or organsresulting from burns, shock, stroke, heart attack or other physicalevents, including complications from surgical or clinical interventions,as a consequence of trauma. Injured soldiers on the battlefield orcivilians at a natural disaster site or injured from a terrorist attackare situations where such treatment may be useful.

The invention applies to protecting, preserving or stabilizing keyorgans such as the heart and brain, other neuronal tissues and cells,renal tissue, lung tissue, muscle tissue, liver and other tissues of thebody.

In one form, the invention provides a method of reducing injury to thecells, tissues or organs of a body following trauma by administering acomposition to the body following trauma including: (i) a glucocorticoidreceptor antagonist; and optionally (ii) another pharmaceutical agent.

The term “trauma” is used herein in its broadest sense and refers to aserious or critical injury, wound or shock to the body. Trauma may becaused by unexpected physical damage (or injury) to the body as a resultof, for example, transport or industrial accidents, birth, surgery,heart attack, stroke, burns, complications due to surgery or othermedical interventions etc. Trauma may result from injury to a body, bothin a hospital or out of hospital. Trauma is often associated with traumamedicine practiced in hospital (such as in hospital emergency rooms),during emergency transport or at out-of-hospital environments where atrauma has occurred, such as domestic or industrial accidents, transportaccidents, the battlefield, and terrorist attacks. In many cases, traumatherapy may also include resuscitation therapy. Exemplary injuriesinclude, for example, burns, shock, stroke, heart attack or otherphysical events, including complications from surgical or clinicalinterventions, as a consequence of trauma. Injured soldiers on thebattlefield or civilians at a natural disaster site or injured from aterrorist attack are situations where such treatment may be useful.

The invention provides a method for treating or preventing infectionrelated to acute or chronic injury or disease wherein said methodcomprises administering to a patient in need of such therapy at leastone glucocorticoid receptor antagonist in a therapeutically effectiveamount.

The activities of GR agonists and their alteration of cellular functionsare variable, depending on complex intracellular molecular signalingthat are cell and tissue specific. Amongst the cells that haveglucocorticoid receptors are stem and progenitor cells of all tissuesand organs of the body.

Thus, binding of such molecules to normative, “in-tissue” stem cells andthe progeny of these stem cells, so-called “transit amplifying”progenitor cells, results in variable, cell and tissue specific effects,inhibitory or enhancing of stem and progenitor cell functions, includingactivation, proliferation, migration and differentiation all of whichare dependent on the tissue/organ in question.

GR antagonists, such as, for example, ORG34517, will thus block theeffects of GR-agonists in tissue specific fashion, enhancingstem/progenitor cell functioning in some, inhibiting it in others.GR-antagonists will have beneficial effects in specific clinicalsettings where regenerative medicine approaches to disease and woundhealing may be of use, including: enhanced post-transplant functioningof autologous stem cell transplants (dependent on tissue of originand/or target tissue).

Attenuation of the peri-surgical effects of catabolic stress hormonesrelated to surgical or other physical traumas (e.g. combat wounds),wherein the acute or chronic injury or disease is selected from thegroup consisting of vascular events, stroke, cardiac arrest, acute limbinfarction accident/battle field trauma, traumatic limb, hip, braininjuries, post-surgical trauma, major orthopedic, thoracic, abdominal,neurological surgeries.

Systemic GR blockade will be inappropriate, but direct application ofORG34517 to site of injury/wounding, either topically (for example, toprevent wound dehiscence) or by direct injection or intravascularinfusion (for visceral organ injuries) will be beneficial.

CNS Injury

The invention provides a method for treating or preventing CNS injury ina patient in need of such treatment, comprising: administering to thepatient therapeutically effective amounts of a GCR (glucocorticoidreceptor) antagonist as exemplified herein, in combination with, forexample, another agent. Conditions suitable for treatment according tothis invention include, for example, seizure disorders, pain syndromes,neurodegenerative diseases (including motor neuron diseases,myelopathies, radiculopathies, and disorders of the sympathetic nervoussystem), dementias, cerebrovascular conditions, movement disorders,brain trauma, cranial nerve disorders, neuropsychiatric disorders, andother disease neuropathies (including viral associated neuropathies,diabetes associated neuropathies, Guillian-Barre syndrome,dysproteinemias, transthyretin-induced neuropathies, and carpal tunnelsyndrome).

As used herein, seizure disorders include complex partial seizures,simple partial seizures, partial seizures with secondary generalization,generalized seizures (including absence, grand mal (tonic clonic),status epilepticus, tonic, atonic, myoclonic), neonatal and infantilespasms, drug-induced seizures, trauma-induced seizures, and febrileseizures, and additional specific epilepsy syndromes such as juvenilemyoclonic epilepsy, Lennox-Gastaut, mesial temporal lobe epilepsy,nocturnal frontal lobe epilepsy, progressive epilepsy with mentalretardation, and progressive myoclonic epilepsy, as well as seizuresassociated with CNS mass lesions.

Pain syndromes include, for example, headaches (e.g., migraine, tension,and cluster), acute pain, chronic pain, neuropathic pain, nociceptivepain, central pain and inflammatory pain, drug-induced neuropathic pain,causalgia, complex regional pain syndrome types I and II, and reflexsympathetic dystrophy (RSDS).

Neurodegenerative diseases include Alzheimer's disease, Parkinson'sDisease, multiple sclerosis, Huntington's Disease, ALS, spinal muscularatrophy, muscular dystrophies prion-related diseases, cerebellar ataxia,Friedrich's ataxia, SCA, Wilson's disease, RP, Gullain Barre syndrome,Adrenoleukodystrophy, Menke's Sx, cerebral autosomal dominantarteriopathy with subcortical infarcts (CADASIL), Charcot Marie Toothdiseases, neurofibromatosis, von-Hippel Lindau, Fragile X, spasticparaplegia, tuberous sclerosis complex, Wardenburg syndrome, spinalmotor atrophies, Tay-Sachs, Sandoff disease, familial spasticparaplegia, myelopathies, radiculopathies, encephalopathies associatedwith trauma, radiation, drugs and infection, and disorders of thesympathetic nervous system (e.g., Shy Drager (familial dysautonomia),diabetic neuropathy, drug-induced and alcoholic neuropathy).

Dementias include Alzheimer's disease, Parkinson's disease, Pick'sdisease, fronto-temporal dementia, vascular dementia, normal pressurehydrocephalus, Huntington's disease, and MCI.

Cerebrovascular conditions amenable to treatment according to thepresent invention include cerebrovascular disease and strokes (e.g.,thrombotic, embolic, thromboembolic, hemorrhagic [including AVM andberry aneurysms], venoconstrictive, and venous).

Included in movement disorders are Parkinson's disease, dystonias,benign essential tremor, tardive dystonia, tardive dyskinesia, andTourette's syndrome.

Brain trauma as used herein includes traumatic brain and spinal cordinjuries as well as brain injuries from radiation.

Cranial nerve disorders include trigeminal neuropathy, trigeminalneuralgia, Menier's syndrome, glossopharangela neuralgia, dysphagia,dysphonia, cranial nerve palsies and Bell's palsy.

Neuropsychiatric disorders include panic syndrome, general anxietydisorder, phobic syndromes of all types, mania, manic depressiveillness, hypomania, unipolar depression, depression, stress disorders,PTSD, somatoform disorders, personality disorders, psychosis, andschizophrenia), and drug dependence/addiction (e.g., alcohol,psychostimulants (e.g., crack, cocaine, speed, meth), opioids, andnicotine), and drug-induced psychiatric disorders.

Other disease neuropathies that may be treated with the compositions andmethods described herein include Guillain-Barre, diabetes associatedneuropathies, dysproteinemias, transthyretin-induced neuropathies,neuropathy associated with HIV, herpes viruses (including herpes zoster)or other viral infection, neuropathy associated with Lyme disease,carpal tunnel syndrome, tarsal tunnel syndrome, amyloid-inducedneuropathies, leprous neuropathy, Bell's palsy, compressionneuropathies, sarcoidosis-induced neuropathy, polyneuritis cranialis,heavy metal induced neuropathy, transition metal-induced neuropathy,drug-induced neuropathy, post-meningitis syndrome, post-polio syndrome,prion diseases, and radiation associated neuropathic syndromes.

Other diseases amenable to treatment with the present invention includefatigue syndromes (e.g., chronic fatigue syndrome and fibromyalgia),ataxic syndromes, olivopontoicerebellar degeneration, striatonigraldegeneration, and axonic brain damage.

The present invention is particularly useful in the treatment ofneuropsychiatric disorders such as depression, agitation, anxiety,seizure disorders such as grand mal seizures, status epilepticus,migraine pain treatment and prophylaxis, Alzheimer's disease,Parkinson's disease, and traumatic brain and spinal cord injury.

Also, the higher doses enabled by the present invention are expected tobe of particular importance for dementias including Alzheimer's disease,Parkinson's disease, and vascular dementia, pain syndromes, includingheadaches and migraines, seizure disorders, movement disorders, andbrain trauma.

Furthermore, the ease of use and convenience of a dosage form provideddeveloped to be delivered at once per day or less frequentadministration at a therapeutically effective quantity from the onset oftherapy is of value in the treatment of dementias including Alzheimer'sdisease and Parkinson's disease, seizure disorders, pain syndromes, andcerebrovascular conditions.

Enhanced Memory and/or Performance

Situational stress can lead to elevated circulating levels of cortisolwhich, in turn, can impair short term memory formation. For example,student exam periods in high school, college, as well as graduate schooland professional certification and licensing exams can lead to suchstress and, therefore, to a self-defeating, cortisol associated deficitin learning that may impair formation of study-based memories and leadto poorer than expected test results. The present invention isparticularly useful in the treatment and/or prevention of short termmemory performance by, for example, single or sequential dosing with aglucocorticoid blocking compound or composition of the invention, beforeand during the examination study period, that will prevent suppressionof short term memory formation and improve study and subsequentexamination performance.

Immunomodulatory Effect

The inventive pharmaceutical composition may additionally contain one ormore auxiliary substances in order to further increase itsimmunomodulatory effect, for example, ORG 34517 (Myvotum) in combinationwith immune system regulators (e.g., IL-1R antagonist) for prevention ofpost-traumatic or disease-associated systemic immunosuppression at highrisk for bacterial infections (e.g., wound infections, pneumonia,colitis, pyelonephritis, hepatic and splenic abscesses) and sepsis. Newcompositions and methods are provided which advantageously employcompounds having a newly defined immune modulating function, or whichhave the ability to mimic that immune modulating function, or acombination of such compounds. For the purposes of the presentdisclosure, the terms “immune mimic,” “immune modulating,” “immunemodulator,” “immune modulation,” “immune control,” “immune inhibition,”“immune suppressor,” and the like, refer in most instances to the newlyidentified cancer cell growth (i.e., proliferation) inhibitory effect ofthe secretory immune system (i.e., dimeric/polymeric IgA and pentamericIgM) that is mediated by a newly identified Poly-Ig receptor orPoly-Ig-like receptor (also classified as an Fc-like receptor), and notto the usual antibody/antigen recognition based immune function of theimmune system. In this context, the terms “immune modulation” or “immuneenhancement” refer especially to the modulation or enhancement of thesecell growth inhibitory immunoglobulins of the secretory immune system.The term “immune mimic” refers to a substance (e.g., tamoxifen) that canfunction in a similar manner to an immunoglobulin inhibitor of cellgrowth. In some instances, however, reference is also made herein to“natural immune inhibition,” “immune enhancer,” “immune modulator,”“immune system,” “immune therapy,” and “immune response,” and the like,in which the conventional meanings of those terms are intended and thecontext so indicates, especially when prior art methods, compounds andcompositions are described. Hereinafter, an indication has been made inappropriate instances whether a conventional definition or the “new”meaning, or both, is intended. A synergistic action of the compounds ofthe invention, e.g., ORG 34517 or a derivative thereof as definedaccording to the present invention, and of an auxiliary substance, whichmay be optionally contained in the inventive pharmaceutical compositionas described above, is preferably achieved thereby. Depending on thevarious types of auxiliary substances, various mechanisms can come intoconsideration in this respect. In general, it is possible to use asauxiliary substance any agent that influences the immune system in themanner of a “danger signal” (LPS, GP96, etc.) or cytokines, such asGM-CSF, which allow for an immune response produced by theimmune-stimulating adjuvant according to the invention to be enhancedand/or influenced in a targeted manner. Particularly preferred auxiliarysubstances are cytokines, such as monokines, lymphokines, interleukinsor chemokines, that further promote the innate immune response, such asIL-1, IL-1 receptor antagonist, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28,IL-29, IL-30, IL-31, IL-32, IL-33, INF-alpha, IFN-beta, INF-gamma,GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.

The inventive pharmaceutical composition can also additionally oralternatively contain any further compound, which is known to beimmune-stimulating due to its binding affinity (as ligands) to humanToll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, or due to its binding affinity (as ligands) to murineToll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12 or TLR13.

Immunoinflammatory Disorder

Another aspect of the present invention is directed to the use of theinventive compound and/or combination as a therapeutic agent for theprophylaxis and/or treatment of immunoinflammatory disorder. The term“immunoinflammatory disorder” encompasses a variety of conditions,including autoimmune diseases, proliferative skin diseases, andinflammatory dermatoses. Immunoinflammatory disorders result in thedestruction of healthy tissue by an inflammatory process, dysregulationof the immune system, and unwanted proliferation of cells. Examples ofimmunoinflammatory disorders are acne vulgaris; acute respiratorydistress syndrome; Addison's disease; allergic rhinitis; allergicintraocular inflammatory diseases, antineutrophil cytoplasmic antibody(ANCA)-associated small-vessel vasculitis; ankylosing spondylitis;arthritis, asthma; atherosclerosis; atopic dermatitis; autoimmunehepatitis; autoimmune hemolytic anemia; autoimmune hepatitis; Behcet'sdisease; Bell's palsy; bullous pemphigoid; cerebral ischemia; chronicobstructive pulmonary disease; cirrhosis; Cogan's syndrome; contactdermatitis; COPD; Crohn's disease; Cushing's syndrome; dermatomyositis;diabetes mellitus; discoid lupus erythematosus; eosinophilic fasciitis;erythema nodosum; exfoliative dermatitis; fibromyalgia; focalglomerulosclerosis; focal segmental glomerulosclerosis; giant cellarteritis; gout; gouty arthritis; graft versus host disease; handeczema; Henoch-Schonlein purpura; herpes gestationis; hirsutism;idiopathic cerato-scleritis; idiopathic pulmonary fibrosis; idiopathicthrombocytopenic purpura; immune thrombocytopenic purpura inflammatorybowel or gastrointestinal disorders, inflammatory dermatoses; lichenplanus; lupus nephritis; lymphomatous tracheobronchitis; macular edema;multiple sclerosis; myasthenia gravis; myositis; nonspecific fibrosinglung disease; osteoarthritis; pancreatitis; pemphigoid gestationis;pemphigus vulgaris; periodontitis; polyarteritis nodosa; polymyalgiarheumatica; pruritus scroti; pruritis/inflammation, psoriasis; psoriaticarthritis; pulmonary histoplasmosis; rheumatoid arthritis; relapsingpolychondritis; rosacea caused by sarcoidosis; rosacea caused byscleroderma; rosacea caused by Sweet's syndrome; rosacea caused bysystemic lupus erythematosus; rosacea caused by urticaria; rosaceacaused by zoster-associated pain; sarcoidosis; scleroderma; segmentalglomerulosclerosis; septic shock syndrome; shoulder tendinitis orbursitis; Sjogren's syndrome; Still's disease; stroke-induced brain celldeath; Sweet's disease; systemic lupus erythematosus; systemicsclerosis; Takayasu's arteritis; temporal arteritis; toxic epidermalnecrolysis; transplant-rejection and transplant-rejection-relatedsyndromes; tuberculosis; type-1 diabetes; ulcerative colitis; uveitis;vasculitis; and Wegener's granulomatosis.

As used herein, “non-dermal inflammatory disorders” include, forexample, rheumatoid arthritis, inflammatory bowel disease, asthma, andchronic obstructive pulmonary disease. By “dermal inflammatorydisorders” or “inflammatory dermatoses” is meant an inflammatorydisorder selected from psoriasis, guttate psoriasis, inverse psoriasis,pustular psoriasis, erythrodermic psoriasis, acute febrile neutrophilicdermatosis, eczema, asteatotic eczema, dyshidrotic eczema, vesicularpalmoplantar eczema, acne vulgaris, atopic dermatitis, contactdermatitis, allergic contact dermatitis, dermatomyositis, exfoliativedermatitis, hand eczema, pompholyx, rosacea, rosacea caused bysarcoidosis, rosacea caused by scleroderma, rosacea caused by Sweet'ssyndrome, rosacea caused by systemic lupus erythematosus, rosacea causedby urticaria, rosacea caused by zoster-associated pain, Sweet's disease,neutrophilic hidradenitis, sterile pustulosis, drug eruptions,seborrheic dermatitis, pityriasis rosea, cutaneous kikuchi disease,pruritic urticarial papules and plaques of pregnancy, Stevens-Johnsonsyndrome and toxic epidermal necrolysis, tattoo reactions, Wellssyndrome (eosinophilic cellulitis), reactive arthritis (Reiter'ssyndrome), bowel-associated dermatosis-arthritis syndrome, rheumatoidneutrophilic dermatosis, neutrophilic eccrine hidradenitis, neutrophilicdermatosis of the dorsal hands, balanitis circumscriptaplasmacellularis, balanoposthitis, Behcet's disease, erythema annularecentrifugum, erythema dyschromicum perstans, erythema multiforme,granuloma annulare, hand dermatitis, lichen nitidus, lichen planus,lichen sclerosus et atrophicus, lichen simplex chronicus, lichenspinulosus, nummular dermatitis, pyoderma gangrenosum, sarcoidosis,subcorneal pustular dermatosis, urticaria, and transient acantholyticdermatosis.

By “proliferative skin disease” is meant a benign or malignant diseasethat is characterized by accelerated cell division in the epidermis ordermis. Examples of proliferative skin diseases are psoriasis, atopicdermatitis, nonspecific dermatitis, primary irritant contact dermatitis,allergic contact dermatitis, basal and squamous cell carcinomas of theskin, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignantkeratosis, acne, and seborrheic dermatitis. As will be appreciated byone skilled in the art, a particular disease, disorder, or condition maybe characterized as being both a proliferative skin disease and aninflammatory dermatosis. An example of such a disease is psoriasis.

Symptoms and signs of inflammation associated with specific conditionsinclude: rheumatoid arthritis:—pain, swelling, warmth and tenderness ofthe involved joints; generalized and morning stiffness;insulin-dependent diabetes mellitus-insulitis; this condition can leadto a variety of complications with an inflammatory component,including:—retinopathy, neuropathy, nephropathy; coronary arterydisease, peripheral vascular disease, and cerebrovascular disease;autoimmune thyroiditis:—weakness, constipation, shortness of breath,puffiness of the face, hands and feet, peripheral edema, bradycardia;multiple sclerosis:—spasticity, blurry vision, vertigo, limb weakness,paresthesias; uveoretinitis:—decreased night vision, loss of peripheralvision; lupus erythematosus:—joint pain, rash, photosensitivity, fever,muscle pain, puffiness of the hands and feet, abnormal urinalysis(hematuria, cylinduria, proteinuria), glomerulonephritis, cognitivedysfunction, vessel thrombosis, pericarditis; scleroderma:—Raynaud'sdisease; swelling of the hands, arms, legs and face; skin thickening;pain, swelling and stiffness of the fingers and knees, gastrointestinaldysfunction, restrictive lung disease; pericarditis; renal failure;other arthritic conditions having an inflammatory component such asrheumatoid spondylitis, osteoarthritis, septic arthritis andpolyarthritis:—fever, pain, swelling, tenderness; other inflammatorybrain disorders, such as meningitis, Alzheimer's disease, AIDS dementiaencephalitis:—photophobia, cognitive dysfunction, memory loss; otherinflammatory eye inflammations, such as retinitis:—decreased visualacuity; inflammatory skin disorders, such as, eczema, other dermatites(e.g., atopic, contact), psoriasis, burns induced by UV radiation (sunrays and similar UV sources):—erythema, pain, scaling, swelling,tenderness; inflammatory bowel disease, such as Crohn's disease,ulcerative colitis:—pain, diarrhea, constipation, rectal bleeding,fever, arthritis; asthma:—shortness of breath, wheezing; other allergydisorders, such as allergic rhinitis:—sneezing, itching, runny noseconditions associated with acute trauma such as cerebral injuryfollowing stroke-sensory loss, motor loss, cognitive loss; heart tissueinjury due to myocardial ischemia:—pain, shortness of breath; lunginjury such as that which occurs in adult respiratory distresssyndrome:—shortness of breath, hyperventilation, decreased oxygenation,pulmonary infiltrates; inflammation accompanying infection, such assepsis, septic shock, toxic shock syndrome:—fever, respiratory failure,tachycardia, hypotension, leukocytosis; other inflammatory conditionsassociated with particular organs or tissues, such as: (i) nephritis(e.g., glomeralonephritis):—oliguria, abnormal urinalysis; (ii) inflamedappendix:—fever, pain, tenderness, leukocytosis; (iii) gout:—pain,tenderness, swelling and erythema of the involved joint, elevated serumand/or urinary uric acid; (iv) inflamed gall bladder:—abdominal pain andtenderness, fever, nausea, leukocytosis; (v) congestive heartfailure:—shortness of breath, rales, peripheral edema; (vi) Type IIdiabetes:—end organ complications including cardiovascular, ocular,renal, and peripheral vascular disease; (vii) lung (pulmonary)fibrosis:—hyperventilation, shortness of breath, decreased oxygenation;(viii) vascular disease, such as atherosclerosis and restenosis:—pain,loss of sensation, diminished pulses, loss of function; and (ix)alloimmunity leading to transplant rejection:—pain, tenderness, fever.

Neurodegenerative Disease

Another aspect of the present invention is directed to the use of theinventive compound and/or combination as a therapeutic agent for theprophylaxis and/or treatment of Neurodegenerative Disease.

The present invention also relates generally to the fields of neurologyand psychiatry and to methods of protecting the cells of a mammaliancentral nervous system from damage or injury. Injuries or trauma ofvarious kinds to the central nervous system (CNS) or the peripheralnervous system (PNS) can produce profound and long-lasting neurologicaland/or psychiatric symptoms and disorders. One form that this can takeis the progressive death of neurons or other cells of the centralnervous system (CNS), i.e., neurodegeneration or neuronal degeneration.

Neuronal degeneration as a result of, for example; Alzheimer's disease,multiple sclerosis, cerebral-vascular accidents (CVAs)/stroke, traumaticbrain injury, spinal cord injuries, degeneration of the optic nerve,e.g., ischemic optic neuropathy or retinal degeneration and othercentral nervous system disorders is an enormous medical and publichealth problem by virtue of both its high incidence and the frequency oflong-term sequelae. Animal studies and clinical trials have shown thatamino acid transmitters (especially glutamate), oxidative stress andinflammatory reactions contribute strongly to cell death in theseconditions. Upon injury or upon ischemic insult, damaged neurons releasemassive amounts of the neurotransmitter glutamate, which is excitotoxicto the surrounding neurons. Glutamate is a negatively charged amino acidthat is an excitatory synaptic transmitter in the mammalian nervoussystem.

Although the concentration of glutamate can reach the millimolar rangein nerve terminals its extracellular concentration is maintained at alow level to prevent neurotoxicity. It has been noted that glutamate canbe toxic to neurons if presented at a high concentration. The term“excitotoxicity” has been used to describe the cytotoxic effect thatglutamate (and other such excitatory amino acids) can have on neuronswhen applied at high dosages.

Patients with injury or damage of any kind to the central (CNS) orperipheral (PNS) nervous system including the retina may benefit fromneuroprotective methods. This nervous system injury may take the form ofan abrupt insult or an acute injury to the nervous system as in, forexample, acute neurodegenerative disorders including, but not limitedto; acute injury, hypoxia-ischemia or the combination thereof resultingin neuronal cell death or compromise. Acute injury includes, but is notlimited to, traumatic brain injury (TBI) including, closed, blunt orpenetrating brain trauma, focal brain trauma, diffuse brain damage,spinal cord injury, intracranial or intravertebral lesions (including,but not limited to, contusion, penetration, shear, compression orlaceration lesions of the spinal cord or whiplash shaken infantsyndrome).

In addition, deprivation of oxygen or blood supply in general can causeacute injury as in hypoxia and/or ischemia including, but not limitedto, cerebrovascular insufficiency, cerebral ischemia or cerebralinfarction (including cerebral ischemia or infarctions originating fromembolic occlusion and thrombosis, retinal ischemia (diabetic orotherwise), glaucoma, retinal degeneration, multiple sclerosis, toxicand ischemic optic neuropathy, reperfusion following acute ischemia,perinatal hypoxic-ischemic injury, cardiac arrest or intracranialhemorrhage of any type (including, but not limited to, epidural,subdural, subarachnoid or intracerebral hemorrhage).

Trauma or injury to tissues of the nervous system may also take the formof more chronic and progressive neurodegenerative disorders, such asthose associated with progressive neuronal cell death or compromise overa period of time including, but not limited to, Alzheimer's disease,Pick's disease, diffuse Lewy body disease, progressive supranuclearpalsy (Steel-Richardson syndrome), multisystem degeneration (Shy-Dragersyndrome), chronic epileptic conditions associated withneurodegeneration, motor neuron diseases (amyotrophic lateralsclerosis), multiple sclerosis, degenerative ataxias, cortical basaldegeneration, ALS-Parkinson's-dementia complex of Guam, subacutesclerosing panencephalitis, Huntington's disease, Parkinson's disease,synucleinopathies (including multiple system atrophy), primaryprogressive aphasia, striatonigral degeneration, Machado-Joseph diseaseor spinocerebellar ataxia type 3 and olivopontocerebellar degenerations,bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy(Kennedy's disease), primary lateral sclerosis, familial spasticparaplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease,Tay-Sach's disease, Sandhoff disease, familial spastic disease,Wohlfart-Kugelberg-Welander disease, spastic paraparesis, progressivemultifocal leukoencephalopathy, familial dysautonomia (Riley-Daysyndrome) or prion diseases (including, but not limited toCreutzfeld-Jakob disease, Gerstmann-Strussler-Scheinker disease, Kurudisease or fatal familial insomnia).

In addition, trauma and progressive injury to the nervous system cantake place in various psychiatric disorders, including but not limitedto, progressive, deteriorating forms of bipolar disorder orschizoaffective disorder or schizophrenia, impulse control disorders,obsessive compulsive disorder (OCD), behavioral changes in temporal lobeepilepsy and personality disorders.

In one preferred embodiment the compounds and/or compositions of theinvention would be used to provide neuroprotection in disordersinvolving trauma and progressive injury to the nervous system in variouspsychiatric disorders. These disorders would be selected from the groupconsisting of; schizoaffective disorder, schizophrenia, impulse controldisorders, obsessive compulsive disorder (OCD) and personalitydisorders.

In addition, trauma and injury make take the form of disordersassociated with overt and extensive memory loss including, but notlimited to, neurodegenerative disorders associated with age-relateddementia, vascular dementia, diffuse white matter disease (Binswanger'sdisease), dementia of endocrine or metabolic origin, dementia of headtrauma and diffuse brain damage, dementia pugilistica or frontal lobedementia, including but not limited to Pick's Disease.

Other disorders associated with neuronal injury include, but are notlimited to, disorders associated with chemical, toxic, infectious andradiation injury of the nervous system including the retina, injuryduring fetal development, prematurity at time of birth, anoxic-ischemia,injury from hepatic, glycemic, uremic, electrolyte and endocrine origin,injury of psychiatric origin (including, but not limited to,psychopathology, depression or anxiety), injury from peripheral diseasesand plexopathies (including plexus palsies) or injury from neuropathy(including neuropathy selected from multifocal, sensory, motor,sensory-motor, autonomic, sensory-autonomic or demyelinatingneuropathies (including, but not limited to Guillain-Barre syndrome orchronic inflammatory demyelinating polyradiculoneuropathy) or thoseneuropathies originating from infections, inflammation, immunedisorders, drug abuse, pharmacological treatments, toxins, trauma(including, but not limited to compression, crush, laceration orsegmentation traumas), metabolic disorders (including, but not limitedto, endocrine or paraneoplastic), Charcot-Marie-Tooth disease(including, but not limited to, type 1a, 1b, 2, 4a or 1-X linked),Friedreich's ataxia, metachromatic leukodystrophy, Refsum's disease,adrenomyeloneuropathy, ataxia-telangiectasia, Djerine-Sottas (including,but not limited to, types A or B), Lambert-Eaton syndrome or disordersof the cranial nerves).

Further indications are cognitive disorders. The term “cognitivedisorder” shall refer to anxiety disorders, delirium, dementia, amnesticdisorders, dissociative disorders, eating disorders, mood disorders,schizophrenia, psychotic disorders, sexual and gender identitydisorders, sleep disorders, somatoform disorders, acute stress disorder,obsessive-compulsive disorder, panic disorder, posttraumatic stressdisorder, specific phobia, social phobia, substance withdrawal delirium,Alzheimer's disease, Creutzfeldt-Jakob disease, head trauma,Huntington's disease, HIV disease, Parkinson's disease, Pick's disease,learning disorders, motor skills disorders, developmental coordinationdisorder, communication disorders, phonological disorder, pervasivedevelopmental disorders, Asperger's disorder, autistic disorder,childhood disintegrative disorder, Rett's disorder, pervasivedevelopmental disorder, attention-deficit/hyperactivity disorder (ADHD),conduct disorder, oppositional defiant disorder, pica, ruminationdisorder, tic disorders, chronic motor or vocal tic disorder, Tourette'sdisorder, elimination disorders, encopresis, enuresis, selective mutism,separation anxiety disorder, dissociative amnesia, depersonalizationdisorder, dissociative fugue, dissociative identity disorder, anorexianervosa, bulimia nervosa, bipolar disorders, schizophreniform disorder,schizoaffective disorder, delusional disorder, psychotic disorder,shared psychotic disorder, delusions, hallucinations, substance-inducedpsychotic disorder, orgasmic disorders, sexual pain disorders,dyspareunia, vaginismus, sexual dysfunction, paraphilias, dyssomnias,breathing-related sleep disorder, circadian rhythm sleep disorder,hypersomnia, insomnia, narcolepsy, dyssomnia, parasomnias, nightmaredisorder, sleep terror disorder, sleepwalking disorder, parasomnia, bodydysmorphic disorder, conversion disorder, hypochondriasis, paindisorder, somatization disorder, alcohol related disorders, amphetaminerelated disorders, caffeine related disorders, cannabis relateddisorders, cocaine related disorders, hallucinogen related disorders,inhalant related disorders, nicotine related disorders, opioid relateddisorders, phencyclidine-related disorder, abuse, persisting amnesticdisorder, intoxication, withdrawal.

The term “bipolar and clinical disorders” shall refer to adjustmentdisorders, anxiety disorders, delirium, dementia, amnestic and othercognitive disorders, disorders usually first diagnosed in infancy(e.g.), childhood, or adolescence, dissociative disorders (e.g.dissociative amnesia, depersonalization disorder, dissociative fugue anddissociative identity disorder), eating disorders, factitious disorders,impulse-control disorders, mental disorders due to a general medicalcondition, mood disorders, other conditions that may be a focus ofclinical attention, personality disorders, schizophrenia and otherpsychotic disorders, sexual and gender identity disorders, sleepdisorders, somatoform disorders, substance-related disorders,generalized anxiety disorder (e.g. acute stress disorder, posttraumaticstress disorder), panic disorder, phobia, agoraphobia,obsessive-compulsive disorder, stress, acute stress disorder, anxietyneurosis, nervousness, phobia, posttraumatic stress disorder,posttraumatic stress disorder (PTSD), abuse, obsessive-compulsivedisorder (OCD), manic depressive psychosis, specific phobias, socialphobia, adjustment disorder with anxious features.

Examples for disorders usually first diagnosed in infancy, childhood, oradolescence are: mental retardation, learning disorders, mathematicsdisorder, reading disorder, disorder of written expression, motor skillsdisorders, developmental coordination disorder, communication disorders,expressive language disorder, phonological disorder, mixedreceptive-expressive language disorder, stuttering, pervasivedevelopmental disorders, Asperger's disorder, autistic disorder,childhood disintegrative disorder, Rett's disorder, pervasivedevelopmental disorder, attention-deficit/hyperactivity disorder (ADHD),conduct disorder, oppositional defiant disorder, feeding disorder ofinfancy or early childhood, pica, rumination disorder, tic disorders,chronic motor or vocal tic disorder, Tourette's syndrome, eliminationdisorders, encopresis, enuresis, selective mutism, separation anxietydisorder, reactive attachment disorder of infancy or early childhood,stereotypic movement disorder.

Examples for substance-related disorders are: alcohol related disorders,amphetamine related disorders, caffeine related disorders, cannabisrelated disorders, cocaine related disorders, hallucinogen relateddisorders, inhalant related disorders, nicotine related disorders,opioid related disorders, psychotic disorder, psychotic disorder,phencyclidine-related disorder, abuse, persisting amnestic disorder,anxiety disorder, persisting dementia, dependence, intoxication,intoxication delirium, mood disorder, psychotic disorder, withdrawal,withdrawal delirium, sexual dysfunction, sleep disorder.

The term “neuroprotection” as used herein shall mean; inhibiting,preventing, ameliorating or reducing the severity of the dysfunction,degeneration or death of nerve cells, axons or their supporting cells inthe central or peripheral nervous system of a mammal, including a human.This includes the treatment or prophylaxis of a neurodegenerativedisease; protection against excitotoxicity or ameliorating the cytotoxiceffect of a compound (for example, a excitatory amino acid such asglutamate; a toxin; or a prophylactic or therapeutic compound thatexerts an immediate or delayed cytotoxic side effect including but notlimited to the immediate or delayed induction of apoptosis) in a patientin need thereof.

The term “a patient in need of treatment with a neuroprotective drug” asused herein will refer to any patient who currently has or may developany of the above syndromes or disorders, or any disorder in which thepatient's present clinical condition or prognosis could benefit fromproviding neuroprotection to prevent the development, extension,worsening or increased resistance to treatment of any neurological orpsychiatric disorder.

The term “treating” or “treatment” as used herein, refers to any indiciaof success in the prevention or amelioration of an injury, pathology orcondition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,pathology, or condition more tolerable to the patient; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; or improving a subject's physical or mentalwell-being. The treatment or amelioration of symptoms can be based onobjective or subjective parameters; including the results of a physicalexamination, neurological examination, and/or psychiatric evaluations.

In some embodiments this invention provides methods of neuroprotection.In certain embodiments, these methods comprise administering atherapeutically effective amount of the composition and/or combinationof the invention to a patient who has not yet developed overt, clinicalsigns or symptoms of injury or damage to the cells of the nervous systembut who may be in a high risk group for the development of neuronaldamage because of injury or trauma to the nervous system or because ofsome known predisposition either biochemical or genetic or the findingof a verified biomarker of one or more of these disorders.

Thus, in some embodiments, the methods and compositions of the presentinvention are directed toward neuroprotection in a subject who is atrisk of developing neuronal damage but who has not yet developedclinical evidence. This patient may simply be at “greater risk” asdetermined by the recognition of any factor in a subject's, or theirfamilies, medical history, physical exam or testing that is indicativeof a greater than average risk for developing neuronal damage.Therefore, this determination that a patient may be at a “greater risk”by any available means can be used to determine whether the patientshould be treated with the methods of the present invention.

Accordingly, in an exemplary embodiment, subjects who may benefit fromtreatment by the methods and the composition and/or combination of thisinvention can be identified using accepted screening methods todetermine risk factors for neuronal damage. These screening methodsinclude, for example, conventional work-ups to determine risk factorsincluding but not limited to: for example, head trauma, either closed orpenetrating, CNS infections, bacterial or viral, cerebrovascular diseaseincluding but not limited to stroke, brain tumors, brain edema,cysticercosis, porphyria, metabolic encephalopathy, drug withdrawalincluding but not limited to sedative-hypnotic or alcohol withdrawal,abnormal perinatal history including anoxia at birth or birth injury ofany kind, cerebral palsy, learning disabilities, hyperactivity, historyof febrile convulsions as a child, history of status epilepticus, familyhistory of epilepsy or any seizure related disorder, inflammatorydisease of the brain including lupis, drug intoxication either direct orby placental transfer, including but not limited to cocaine poisoning,parental consanguinity, and treatment with medications that are toxic tothe nervous system including psychotropic medications.

The determination of which patients may benefit from treatment with aneuroprotective drug in patients who have no clinical signs or symptomsmay be based on a variety of “surrogate markers” or “biomarkers”.

As used herein, the terms “surrogate marker” and “biomarker” are usedinterchangeably and refer to any anatomical, biochemical, structural,electrical, genetic or chemical indicator or marker that can be reliablycorrelated with the present existence or future development of neuronaldamage. In some instances, brain-imaging techniques, such as computertomography (CT), magnetic resonance imaging (MRI) or positron emissiontomography (PET), can be used to determine whether a subject is at riskfor neuronal damage. Suitable biomarkers for the methods of thisinvention include, but are not limited to: the determination by MRI, CTor other imaging techniques, of sclerosis, atrophy or volume loss in thehippocampus or overt mesial temporal sclerosis (MTS) or similar relevantanatomical pathology; the detection in the patient's blood, serum ortissues of a molecular species such as a protein or other biochemicalbiomarker, e.g., elevated levels of ciliary neurotrophic factor (CNTF)or elevated serum levels of a neuronal degradation product; or otherevidence from surrogate markers or biomarkers that the patient is inneed of treatment with a neuroprotective drug.

It is expected that many more such biomarkers utilizing a wide varietyof detection techniques will be developed in the future. It is intendedthat any such marker or indicator of the existence or possible futuredevelopment of neuronal damage, as the latter term is used herein, maybe used in the methods of this invention for determining the need fortreatment with the compounds and methods of this invention.

A determination that a subject has, or may be at risk for developing,neuronal damage would also include, for example, a medical evaluationthat includes a thorough history, a physical examination, and a seriesof relevant bloods tests. It can also include an electroencephalogram(EEG), CT, MRI or PET scan. A determination of an increased risk ofdeveloping neuronal damage or injury may also be made by means ofgenetic testing, including gene expression profiling or proteomictechniques. For psychiatric disorders that may be stabilized or improvedby a neuroprotective drug, e.g., bipolar disorder, schizoaffectivedisorder, schizophrenia, impulse control disorders, etc. the above testsmay also include a present state exam and a detailed history of thecourse of the patients symptoms such as mood disorder symptoms andpsychotic symptoms over time and in relation to other treatments thepatient may have received over time, e.g., a life chart. These and otherspecialized and routine methods allow the clinician to select patientsin need of therapy using the methods and fonnulations of this invention.In some embodiments of the present invention compujds and/or compostionssuitable for use in the practice of this invention will be administeredeither singly or concomitantly with at least one or more other compoundsor therapeutic agents, e.g., with other neuroprotective drugs orantiepileptic drugs, anticonvulsant drugs. In these embodiments, thepresent invention provides methods to treat or prevent neuronal injuryin a patient. The method includes the step of; administering to apatient in need of treatment, an effective amount of the compunds and/orcompositionss disclosed herein in combination with an effective amountof one or more other compounds or therapeutic agents that have theability to provide neuroprotection or to treat or prevent seizures orepileptogenesis or the ability to augment the neuroprotective effects ofthe compounds of the invention.

As used herein the term “combination administration” of a compound,therapeutic agent or known drug with the combination of the presentinvention means administration of the drug and the one or more compoundsat such time that both the known drug and/or combination will have atherapeutic effect. In some cases this therapeutic effect will besynergistic. Such concomitant administration can involve concurrent(i.e. at the same time), prior, or subsequent administration of the drugwith respect to the administration of the composition and/or combinationof the present invention. A person of ordinary skill in the art wouldhave no difficulty determining the appropriate timing, sequence anddosages of administration for particular drugs of the present invention.

The said one or more other compounds or therapeutic agents may beselected from compounds that have one or more of the followingproperties: antioxidant activity; NMDA receptor antagonist activity,augmentation of endogenous GABA inhibition; NO synthase inhibitoractivity; iron binding ability, e.g., an iron chelator; calcium bindingability, e.g., a Ca (II) chelator; zinc binding ability, e.g., a Zn (II)chelator; the ability to effectively block sodium or calcium ionchannels, or to open potassium or chloride ion channels in the CNS of apatient.

Heart and Vascular Disease

Another aspect of the present invention is directed to the use of theinventive compound and/or combination as a therapeutic agent for theprophylaxis and/or treatment of heart disease. Heart disease is ageneral term used to describe many different heart conditions. Forexample, coronary artery disease, which is the most common heartdisease, is characterized by constriction or narrowing of the arteriessupplying the heart with oxygen-rich blood, and can lead to myocardialinfarction, which is the death of a portion of the heart muscle. Heartfailure is a condition resulting from the inability of the heart to pumpan adequate amount of blood through the body. Heart failure is not asudden, abrupt stop of heart activity but, rather, typically developsslowly over many years, as the heart gradually loses its ability to pumpblood efficiently. Risk factors for heart failure include coronaryartery disease, hypertension, valvular heart disease, cardiomyopathy,disease of the heart muscle, obesity, diabetes, and/or a family historyof heart failure.

Examples of cardiovascular diseases and disorders are: aneurysm, stableangina, unstable angina, angina pectoris, angioneurotic edema, aorticvalve stenosis, aortic aneurysm, arrhythmia, arrhythmogenic rightventricular dysplasia, arteriosclerosis, arteriovenous malformations,atrial fibrillation, Behcet syndrome, bradycardia, cardiac tamponade,cardiomegaly, congestive card iomyopathy, hypertrophic cardiomyopathy,restrictive cardiomyopathy, carotid stenosis, cerebral hemorrhage,Churg-Strauss syndrome, diabetes, Ebstein's Anomaly, Eisenmengercomplex, cholesterol embolism, bacterial endocarditis, fibromusculardysplasia, congenital heart defects, heart diseases, congestive heartfailure, heart valve diseases, heart attack, epidural hematoma,hematoma, subdural, Hippel-Lindau disease, hyperemia, hypertension,pulmonary hypertension, cardiac hypertrophy, left ventricularhypertrophy, right ventricular hypertrophy, hypoplastic left heartsyndrome, hypotension, intermittent claudication, ischemic heartdisease, Klippel-Trenaunay-Weber syndrome, lateral medullary syndrome,long QT syndrome mitral valve prolapse, moyamoya disease, mucocutaneouslymph node syndrome, myocardial infarction, myocardial ischemia,myocarditis, pericarditis, peripheral vascular diseases, phlebitis,polyarteritis nodosa, pulmonary atresia, Raynaud disease, Sneddonsyndrome, superior vena cava syndrome, syndrome X, tachycardia,Takayasu's arteritis, hereditary hemorrhagic telangiectasia,telangiectasis, temporal arteritis, tetralogy of Fallot, thromboangiitisobliterans, thrombosis, thromboembolism, tricuspid atresia, varicoseveins, vascular diseases, vasculitis, vasospasm, ventricularfibrillation, Williams syndrome, peripheral vascular disease, varicoseveins and leg ulcers, deep vein thrombosis, Wolff-Parkinson-Whitesyndrome.

Vascular diseases are often the result of decreased perfusion in thevascular system or physical or biochemical injury to the blood vessel.

Peripheral vascular disease (PVD) is defined as a disease of bloodvessels often encountered as narrowing of the vessels of the limbs.There are two main types of these disorders, functional disease whichdoesn't involve defects in the blood vessels but rather arises fromstimuli such as cold, stress, or smoking, and organic disease whicharises from structural defects in the vasculature such asatherosclerotic lesions, local inflammation, or traumatic injury. Thiscan lead to occlusion of the vessel, aberrant blood flow, and ultimatelyto tissue ischemia.

One of the more clinically significant forms of PVD is peripheral arterydisease (PAD). PAD is often treated by angioplasty and implantation of astent or by artery bypass surgery. Clinical presentation depends on thelocation of the occluded vessel. For example, narrowing of the arterythat supplies blood to the intestine can result in severe postprandialpain in the lower abdomen resulting from the inability of the occludedvessel to meet the increased oxygen demand arising from digestive andabsorptive processes. In severe forms the ischemia can lead tointestinal necrosis. Similarly, PAD in the leg can lead to intermittentpain, usually in the calf, that comes and goes with activity. Thisdisorder is known as intermittent claudication (IC) and can progress topersistent pain while resting, ischemic ulceration, and even amputation.

Peripheral vascular disease is also manifested in atheroscleroticstenosis of the renal artery, which can lead to renal ischemia andkidney dysfunction.

One disease in which vascular diseases and their complications are verycommon is diabetes mellitus. Diabetes mellitus causes a variety ofphysiological and anatomical irregularities, the most prominent of whichis the inability of the body to utilize glucose normally, which resultsin hyperglycemia. Chronic diabetes can lead to complications of thevascular system which include atherosclerosis, abnormalities involvinglarge and medium size blood vessels (macroangiopathy) and abnormalitiesinvolving small blood vessels (microangiopathy) such as arterioles andcapillaries.

Patients with diabetes mellitus are at increased risk of developing oneor more foot ulcers as a result of established long-term complicationsof the disease, which include impaired nerve function (neuropathy)and/or ischemia. Local tissue ischemia is a key contributing factor todiabetic foot ulceration.

In addition to large vessel disease, patients with diabetes sufferfurther threat to their skin perfusion in at least two additional ways.First, by involvement of the non-conduit arteries, which aredetrimentally affected by the process of atherosclerosis, and secondly,and perhaps more importantly, by impairment of the microcirculatorycontrol mechanisms (small vessel disease). Normally, when a body partsuffers some form of trauma, the body part will, as part of the body'shealing mechanism, experience an increased blood flow. When small vesseldisease and ischemia are both present, as in the case of many diabetics,this natural increased blood flow response is significantly reduced.This fact, together with the tendency of diabetics to form blood clots(thrombosis) in the microcirculatory system during low levels of bloodflow, is believed to be an important factor in ulcer pathogenesis.

Neuropathy is a general term which describes a disease process whichleads to the dysfunction of the nervous system, and is one of the majorcomplications of diabetes mellitus, with no well-established therapiesfor either its symptomatic treatment or for prevention of progressivedecline in nerve function.

The thickening and leakage of capillaries caused by diabetes primarilyaffect the eyes (retinopathy) and kidneys (nephropathy). The thickeningand leakage of capillaries caused by diabetes are also associated withskin disorders and disorders of the nervous system (neuropathy).

The eye diseases associated with diabetes are nonproliferative diabeticretinopathy, proliferative diabetic retinopathy, diabetic maculopathy,glaucoma, cataracts and the like.

Other diseases, although not known to be related to diabetes are similarin their physiological effects on the peripheral vascular system. Suchdiseases include Raynaud syndrome, CREST syndrome, autoimmune diseasessuch as erythematosis, rheumatoid disease, and the like.

As used herein, the term “peripheral vascular diseases” comprises anyperipheral vascular disease including peripheral and autonomicneuropathies. Examples of “peripheral vascular disease” includeperipheral arterial disease, such as chronic arterial occlusionincluding arteriosclerosis, arteriosclerosis obliterans andthromboangiitis obliterans (Buerger's disease), macroangiopathy,microangiopathy, diabetes mellitus, thrombophlebitis, phlebemphraxis,Raynaud's disease, Raynaud's syndrome, CREST syndrome, health hazard dueto vibration, Sudeck's syndrome, intermittent claudication, cold sensein extremities, abnormal sensation in extremities, sensitivity to thecold, Meniere's disease, Meniere's syndrome, numbness, lack ofsensation, anesthesia, resting pain, causalgia (burning pain),disturbance of peripheral circulation function, disturbance of nervefunction, disturbance of motor function, motor paralysis, diabeticperipheral circulation disorder, lumbar spinal canal stenosis, diabeticneuropathy, shock, autoimmune disease such as erythematosis, rheumatoiddisease and rheumatoid arthritis, autonomic neuropathy, diabeticautonomic neuropathy, autonomic imbalance, orthostatic hypotension,erectile dysfunction, female sexual dysfunction, retrograde ejaculation,cystopathy, neurogenic bladder, defective vaginal lubrication, exerciseintolerance, cardiac denervation, heat intolerance, gustatory sweating,diabetic complication, hyperglycemia, hypoglycemia unawareness,hypoglycemia unresponsiveness; glaucoma, neovascular glaucoma, cataract,retinopathy, diabetic retinopathy, diabetic maculopathy, occlusion ofretinal artery, obstruction of central artery of retina, occlusion ofretinal vein, macular edema, aged macular degeneration, aged disciformmacular degeneration, cystoid macular edema, palpebral edema, retinaledema, chorioretinopathy, neovascular maculopathy, uveitis, iritis,retinal vasculitis, endophthalmitis, panophthalmitis, metastaticophthalmia, choroiditis, retinal pigment epithelitis, conjunctivitis,cyclitis, scleritis, episcleritis, optic neuritis, retrobulbar opticneuritis, keratitis, blepharitis, exudative retinal detachment, cornealulcer, conjunctival ulcer, chronic nummular keratitis, Thygesonkeratitis, progressive Mooren's ulcer, damage of skin, skin ulcerincluding foot ulcer, diabetic ulcer, burn ulcer, lower leg ulcer,postoperative ulcer, traumatic ulcer, ulcer after herpes zoster,radiation ulcer, drug induced ulcer, frostbite (cold injury), chilblain,gangrene and sudden gangrene, angina pectoris/variant angiitis, coronaryarteriosclerosis (chronic ischemic heart disease, asymptomatic ischemicheart disease, arteriosclerotic cardiovascular disease), myocardialinfarction, heart failure, congestive heart failure and painlessischemic heart disease, pulmonary edema, hypertension, pulmonaryhypertension; portal hypertension, diabetic nephropathy, decubitus,renal failure.

Cortisol

The estimated daily cortisol production rate in normal subjects variesbetween 4-15 mg/m² per day or, according to more recent studies between9 and 11 mg/m.sup.2 per day. In order to describe the 24-hour variationin serum cortisol levels adequately, the day may be divided into, forexample, four phases. Phase 1 is a 6-hours period of minimal secretoryactivity 4 h before and 2 h after onset of sleep. Phase 2 refers to the3rd to 5th hours of sleep when there is a preliminary nocturnalsecretory episode. Phase 3 is a 4-hour main secretory phase during thelast 3 h of sleep and the first hour after wakening. Phase 4 is an11-hour phase of intermittent secretory activity when there is a slowdecline in serum levels of cortisol.

In a study by Mah et al. (Clinical Endocrinology (2004) 61, 367-375) thecircadian rhythm of serum cortisol of normal subjects is described. Peaklevels of about 400-800 mmol/l, about 150-300 mmol/l and about 150mmol/l are observed at about 6 am, 2 pm and 9 pm, respectively, and thelowest level is about midnight. In this study it is observed that theendogenous cortisol levels reach their highest levels within 30 minutesafter wake-up. In order to mimic the circadian rhythm, Mah et al.recommend a thrice-daily treatment regimen of hydrocortisone, the firstdose taken in the fasted state and delaying the breakfast 1-3 hours andthe other two doses taken 15-60 min before food. A trice-daily regimenis also recommended in a recent review by Czock et al. (Clin.Pharmacokinet (2005) 44, 61-98) due to the short half-life ofhydrocortisone, and for prednisolone a twice-daily regimen is preferredover a once-daily regimen.

Cortisol Test

The absence of rapid response and inexpensive testing for cortisol has,heretofore, prevented the linking of GCR antagonists (e.g., ORG 34517)to a cortisol pre-test for entry into clinical trials for GCRantagonists and will inhibit the ability to select the patients mostlikely to receive the benefit of treatment with the compounds whenavailable for clinical use. The invention provides the pairing of anaffordable, real-time cortisol test (e.g., PopTest Cortisol) which willenable the successful completion of clinical trials for this class ofdrugs as well as form the basis for their future, anticipatedtherapeutic use(s).

Conditions that may be treated using, for example, a linked salivarycortisol quantification test and GCR antagonist (e.g., ORG 34517) systeminclude, but are not limited to the following:

Major Depressive Disorder (MDD)

MDD is a psychiatric disorder which has a lifetime prevalence of around8%. One of the most consistent findings in psychiatry is that patientswith major depression present with alterations in thehypothalamic-pituitary-adrenal (HPA) axis. A significant percentage ofdepressed patients exhibit hypersecretion cortisol, as manifested byelevated plasma, cerebrospinal fluid, and salivary concentrations ofcortisol and increased urinary free cortisol. In addition, manydepressed patients exhibit a clear inability to switch off endogenouscortisol release following exogenous challenge with the potent syntheticglucocorticoid dexamethasone (the so-called dexamethasonenon-suppressors) (Gold P. W., et al., Clinical and biochemicalmanifestations of depression: relation to neurobiology of stress. NewEngland J. Med. 319, 413-420, 1988). This ‘sub-group’ of severelycompromised patients are most often the ones in whom depression becomesa life-threatening illness that warrants hospitalization.

Other abnormalities of the HPA axis found in depressed patients areincreased cortisol response to corticotrophin, a blunted corticotrophinresponse to CRH (corticotrophin releasing hormone), and adrenal andpituitary enlargement (for a review see Holsboer, F. and Barden, N.:Endocrine Reviews 1996, 17, 187-205). These observations have beeninterpreted to suggest a causal relationship between disturbedfunctioning of the HPA axis and the pathology of depression (Murphy, B.E. P. J. of Steroid Biochem. and Mol. Biol. 1991, 38, 537-559).Therapeutic efficacy of classical antidepressants has been shown to bepreceded by or to coincide with restoration of the disturbed HPA axis indepression (Holsboer and Barden, 1996, supra). It has been postulatedthat any intervention which can restore this HPA dysfunction may haveantidepressant potential.

One type of such intervention, studies of which support the impressionthat HPA-axis functioning and high circulating cortisol is a majorinstigator of major depression is the administration of glucocorticoidsynthesis inhibitors, as has been shown in patients suffering fromCushing's Syndrome, which is a condition in which high cortisol levelsare reported as a result of adrenal gland malfunction (due to apituitary tumour or a secondary tumour, both producing the cortisolsecretagogue ACTH). The depressive symptoms associated with Cushing'sdisappear relatively quickly with the return of cortisol levels tonormal. Such treatment may involve removal of the offending tumour ortreatment with cortisol synthesis inhibitors such as metyrapone,ketoconozole, or aminoglutethimide (Murphy, B. E. P., Steroids andDepression. J. Steroid Biochem & Mol. Biol. 38, 537-558, 1991).Similarly, relatively recent clinical trials have demonstrated thatcortisol synthesis inhibitors can be used to ameliorate depressivesymptoms in severe, treatment-resistant non-Cushing depressives (Murphy,B. E. P., Can. J. Psych. 43, 279-286, 1998; see also U.S. Pat. No.4,814,333 (Ravaris, C. L.)).

Another type of intervention is the use of direct GCR antagonists, whichhave much more specific pharmacological effects as compared to synthesisinhibitors and which may help restore HPA activity Small scale pilotclinical studies have been conducted in order to study theantidepressant activity of the non-selective glucocorticoid receptorantagonist RU 486 (mifepristone; Murphy, B. E. P. et al. J. Psychiat.Neurosc. 18, 209-213, 1993). More recently (Nemeroff, C., RemeronScientific Expert Meeting, Budapest, March 29-Apr. 1, 2001) it wasdemonstrated in a Phase IIB continuation of this study, that both thenumber of responders as well as the efficacy of the psychosis treatmentincreased with increasing daily dose of mifepristone as measured by thechange in Brief Psychiatric Rating Scale (50 mg-33% change; 600 mg-40%change and 1200 mg-52% change). These data indicate that a higher doseof glucocorticoid receptor antagonist is correlated with a higherclinical efficacy. Non-response to standard treatments, however, reachlevels as high as 50%. (Connolly K R, Drugs. 2011; 71: 43-64.)Frequently, extra interventions are necessary to get patients to achieveremission. Various augmentation and combination strategies have beendescribed in the literature for difficult to treat major MMD patients.

Use of an HPA-axis modulating drug in these patients has not beenstudied in spite of the fact that there is clear evidence that at leasta sub-group of MDD patients have significant HPA-axis dysfunction, asnoted above. Biological symptoms, indicative of excessive activity ofthe HPA-axis, have been reported with great consistency. In parallel,there is a body of evidence suggesting that there is an associationbetween HPA-axis functioning and treatment response, where high HPA-axisactivation at baseline, or post-treatment, is associated with a poorerresponse to SSRI treatment or a higher relapse risk.

Preclinical studies indicate that HPA-axis dysfunction of the type seenin affective disorders can attenuate the neurochemical effects of aselective serotonin re-uptake inhibitor (SSRI) antidepressant.Conversely, in animals with normal HPA axis function, co-administrationof GR antagonists augmented the neurochemical effects of an S SRI. Thesedata provide a mechanistic underpinning of the GR antagonistaugmentation strategy, and moreover indicate that the strategy may proveefficacious in patients both with and without HPA axis dysfunction.

Small scale pilot clinical studies were conducted in order to study theantidepressant activity of the non-selective glucocorticoid receptorantagonist RU 486 (mifepristone; Murphy, B. E. P. et al. J. Psychiat.Neurosc. 18, 209-213, 1993). A double blind, 4 week, paroxetinecontrolled study of ORG 34517 in depressed patients was carried out.Paroxetine is a selective serotonin re-uptake inhibitor which isrecognized as an effective antidepressant for major depression. Patientswere selected which had a primary depressive disorder fulfilling thediagnostic criteria of a MDD as defined by the DSM-IV for recurrent(296.3) episodes, and who had a severity of depression which resulted ina total score of at least 22 on the HAMD-21 (Hamilton Rating Scale forDepression; see Hamilton, M. “A rating scale for depression.” J. Neurol.Neurosurg. Psychiat. 1960, 23, 56-62) scale at baseline. Patients had anepisode of depression which had lasted at least 2 weeks before baseline.

In this study, patients were randomly allocated to one of threetreatment groups. Group I patients (50 patients) received 2 capsuleswith 75 mg of ORG 34517 and one placebo (total daily dose 150 mg) forthe first 2 weeks and 2 capsules with 75 mg ORG 34517 and 1 capsule with150 mg (total daily dose 300 mg) the next 2 weeks; Group II patients (46patients) received 3 capsules with 150 mg ORG 34517 (total daily dose450 mg) in the first 2 weeks and 4 capsules of ORG 34517 (total dailydose 600 mg) in the next 2 weeks; Group III patients (44 patients)received 2 capsules with 10 mg paroxetine and one placebo capsule (totaldaily dose 20 mg) for the first 2 weeks, followed by 2 capsules of 10 mgand one capsule of 20 mg paroxetine (total daily dose 40 mg) in the next2 weeks. Medication was administered orally in the morning. Efficacyassessment was done on days 4, 7, 10, 14, 21, 28 and 35 by using the21-item HAMD scale.

Thus, GCR antagonist therapy could prove a useful mechanism fortreatment of selected individuals who fail to respond to currentanti-depressant therapies such as SSRIs, providing a way to enhanceresponsiveness or as an alternate means of achieving a maintainedeuthymia.

Psychotic Depression

Psychotic major depression has long been recognized as a distinctpsychiatric illness, having both psychotic and depressive components ina differential diagnosis. Psychotic major depression is very common. Ithas been estimated that twenty five percent of depressed patientsadmitted to the hospital have psychotic major depression (Coryell (1984)J. Nerv. Ment. Dis. 172:521). Like major depression, psychoticdepression is often also a result of high circulating cortisol levels.Various evidence supports this concept. Psychosis has been associatedwith Cushing's syndrome (Gerson (1985) Can. J. Psychiatry 30:223-224;Saad (1984) Am. J. Med. 76:759-766). A GR antagonist has been used totreat acute psychiatric disturbances secondary to Cushing's syndrome.One study showed that a relatively high dose of such a GR antagonist(400 to 800 mg per day) was useful in rapidly reversing acute psychosisin patients with severe Cushing's′ Syndrome due to adrenal cancers andectopic secretion of ACTH from lung cancer (Van der Lely (1991) Ann.Intern. Med. 114:143; Van der Lely (1993) Pharmacy World & Science15:89-90; Sartor (1996) supra). Relatively high dose mifepristone, inthe range of 8-12 mg/kg/day, over a relatively short period of time (4days), was also shown to be effective in the treatment of psychosisassociated with psychotic major depression (International PatentApplication WO 99/17779; Schatzberg and Belanoff).

Surgery-Associated Immune Suppression in the Elderly.

In healthy, young to middle aged subjects suffering from stress, thereis a physiological balance between pro-inflammatory andanti-inflammatory mediators. In the elderly, the immune response isblunted as a result of the decline in several components of the immunesystem (immune senescence) and a shifting to a chronic pro-inflammatorystatus (the so-called “inflammaging” effect (Butcher and Lord, (2004)Aging Cell, pp. 151-160). As production of cortisol remains reasonablyconstant with age, whereas summed levels of DHEA and DHEAS decreasegradually from the third decade, reaching 10-20% of their maximum by theeighth decade, Butcher and Lord (2004, supra) propose a model for ageand stress, in which the age-related increase in the ratio of cortisolto DHEAS, combined with an elevated cortisol release during stress,leads to a significant reduction of immunity in aging subjects. This isproposed to explain that aging subjects are far more prone to infectionsunder conditions of stress. (Butcher and Lord (2004, supra); Butcher etal. (2005, Aging Cell 5, pp. 319-324). The present invention relates tothe use of a GCR antagonist for the prevention or treatment ofinfections or infectious conditions, in an aging patient, such as ahuman subject. The beneficial effects of said GCR antagonists may beexplained on the basis of their correcting influence on thecortisol/DHEA(S) ratio. It is believed that the effect in selectedsubjects, found to have high circulating cortisol by a saliva test asprovided for by this invention, can be explained by the unbalancedimmunosuppressive role of the increased cortisol/DHEAS ratio in the agedgroup in comparison to the balanced influence of cortisol and DHEAS onthe immune system in normal subjects.

The meaning of the term ‘aging subject’ or ‘aged subject’ will be wellunderstood in the context of the use according to this invention.Although it is not linked to an exact lower age limit this generalnotion refers in the human situation usually to a person of at least 55years old, but it is more clear with a lowest age limit set at 60, 65,70 or 75 years.

In the context of the invention, the infection or infectious conditioncan be caused by any of several agents, e.g., by bacteria, by viruses orby fungi. Also in the context of the present invention, the expression“infectious conditions” means silent or subclinical infections as wellas conditions not resulting in a manifest infectious disease, but inwhich at least one parameter associated with an infectious disease, suchas the white blood (e.g., neutrophil, basophil or eosinophil) cellcounts or the level of some antibodies or some cytokines is higher thannormal. Normal values are known to the expert and may be found instandard medical manuals.

Particular uses according to the invention relate to aging subjectssuffering from an infection or an infectious condition concomitant tostress resulting from a trauma. The invention particularly relates touses wherein the subject suffers from the consequences of a bonefracture and/or bone surgery, either for such injury or for jointreplacement for osteoarthritis or rheumatoid arthritis. The inventionalso relates to uses wherein the subject suffers from an infection or aninfectious condition concomitant to psychological stress, particularlyacute emotional stress.

Post-Traumatic Stress Disorder (PTSD)

PTSD is a severe anxiety disorder that can develop after exposure to anyevent that results in psychological trauma. This event may involve thethreat of death to oneself or to someone else, or to one's own orsomeone else's physical, sexual, or psychological integrity,overwhelming the individual's ability to cope. As an effect ofpsychological trauma, PTSD is less frequent and more enduring than themore commonly seen acute stress response. Diagnostic symptoms for PTSDinclude re-experiencing the original trauma(s) through flashbacks ornightmares, avoidance of stimuli associated with the trauma, andincreased arousal, such as difficulty falling or staying asleep, anger,and hyper-vigilance. Formal diagnostic criteria (both DSM-IV-TR andICD-9) require that the symptoms last more than one month and causesignificant impairment in social, occupational, or other important areasof functioning. (Diagnostic and statistical manual of mental disorders:DSM-IV. American Psychiatric Association. 1994. Washington, D.C.:American Psychiatric Association.) PTSD displays biochemical changes inthe brain and body that differ from other psychiatric disorders such asmajor depression. Abundant evidence suggests derangement of HPA-axisphysiology in individuals diagnosed with PTSD, though the nature of thederangements is variable: some have low cortisol, some have normallevels, others have high levels of cortisol and for some, levels may benormal, but circadian rhythm is lost. It is postulated that thesereflect different baseline mechanisms, but that when cortisol is high,either in a sustained way through the day or by loss of circadian rhythmwith elevated night time levels, it is likely to be an importantcomponent of the clinical symptomatology (Lindley S E, et al. Basal anddexamethasone suppressed salivary cortisol concentrations in a communitysample of patients with posttraumatic stress disorder. Biol. Psychiatry2004; 55: 940-5). In such patients, determined by salivary cortisoltesting, administration of a GCR antagonist is expected to betherapeutic or beneficial for the symptoms of PTSD.

Prevention of Weight Gain in Patients Using Anti-Psychotic andAnti-Depressant Medications

Anti-psychotic and some anti-depressant medications (e.g., SSRIs) areamongst the most important tools for treating psychiatric conditions ofall kinds. However, management of patients on who take many of thesemedications for chronic, long term disease is made difficult by theirsignificant side effect profiles. One of the most important of these isweight gain and the attendant metabolic syndrome that follows. Forexample, it is estimated that 40-80% of patients who are under chronicanti-psychotic administration experience substantial weight gain, oftenexceeding 20% or more over their ideal body weights (Umbricht et al. JClin. Psychiatry 1994; 55: 157-160; Khan A Y, et al. J Psychiatr Pract.2010; 16: 289-96; Pramyothin P, Khaodhiar L. Curr Opin EndocrinolDiabetes Obes. 2010; 17: 460-6.; Rummel-Kluge C et al. Schizophr Res.2010; 123: 225-33). Such weight gain is one of the most common causes ofpoor compliance with anti-psychotic and anti-depressant regimens and,therefore, of long term failure of therapy. Furthermore, anti-psychoticmedications specifically are commonly associated with development ofinsulin resistance and metabolic syndrome (with development of type 2diabetes mellitus and hyper/dyslipidemia states) and the potentially andsignificantly increased risks for cardiovascular disease; theseconditions are of tremendous medical consequence for patients who arethereby caught in a “can't live with them, can't live without them”treatment scenario. While weight gain is potentially seen with allanti-psychotic medications, they are particularly common and tend tomore severe with the newer or “atypical” AP drugs (Allison et al. Am JPsychiatry 1999; 156:1686-1696; Rummel-Kluge C et al. Schizophr Res.2010; 123: 225-33).

Elevations in cortisol are associated with changes in body fat andinsulin resistance. Several years ago, in a proof of principle clinicalexperiment, it was reported that one GCR anatogonist (mifepristone) wasa highly effective treatment for multiple medical complications in apatient with Cushing's disease whose illness had not responded tosurgery and radiation, including reversal of insulin dependent diabetes:the patient was able to stop insulin within a month (Chu et al., J.Clin. Endocrinol. Metab. 2001; 86, 3568-3573.). These data suggest thata GCR antagonist could be useful for blocking and reversing the insulinresistance and weight changes seen in some patients treated withatypical antipsychotic agents. To this end, this compound was tested inrats who had olanzapine-induced weight gain and increases in abdominalfat; reversal of weight gain was seen and reduction of abdominal fat wasobtained (Beebe et al. Behav. Brain Res. 2006; 171, 225-229). A clinicaltrial with this compound then confirmed this benefit in humans with a 2week study of 600 mg/day of mifepristone that reduced olanzapine-inducedweight gain in 57 non-overweight healthy males with Body Mass Indicesless than 25 (Gross et al., Adv Ther. 2009; 26: 959-69.). Thus, GCRantagonist therapy could prove a useful mechanism to target in treatingpsychotic patients with atypical antipsychotic agents.

Cushing's Syndrome

Cushing's Syndrome is a set of conditions in which high levels ofcirculating cortisol or other GCR agonists cause a set of seriouslydebilitating and sometimes life threatening signs and symptomsincluding, but not limited to, psychiatric disturbances (e.g. anxiety,depression, psychosis), immunosuppression, insulin resistance andmetabolic syndrome, skin conditions, hypertension and osteoporosis.Endogenous cortisol may be produced by ACTH-secreting, benign ormalignant tumors of the pituitary gland (“Cushing's Disease”) or of theadrenal cortex. These are rare conditions and therefore Cushing'sSyndrome is considered an “orphan disease.”

A proof of concept trial using RU486 to treat patients withtumor-related Cushing's Syndrome demonstrated efficacy in remittingsymptoms such as glucose metabolic abnormalities (i.e., glucoseintolerance; (group 1) and hypertension (group 2). Statisticallysignificant improvement was achieved for both groups: with 60%responding in the glucose intolerant group and 43% in the hypertensivegroup (Corcept Therapeutics Press Release Dec. 22, 2010). Thus, GCRantagonist therapy can be expected to provide clinical benefits forpatients with Cushing's Syndrome administered prior to tumor surgery toimprove surgical outcomes and/or post-surgery to mitigate symptoms inpatients for whom surgical cure is not achievable.

In addition, GCR antagonist therapy can be expected to provide clinicalbenefits for patients, for example, in hospitals, nursing homes,nurseries, daycares, schools, work environments, public transportation,healthcare settings, psychiatric institutions, and long-term nursingfacilities.

Diagnostic Systems and Kits

A diagnostic kit may comprise some or all of the followingcomponents: 1) one or more standards comprised of one or more of thebiomarker(s) of the invention, such as cortisol; 2) a ligand, such as anantibody or a plurality of antibodies, that are specific for thebiomarker(s) that are to be assayed for using the kit; 3) writteninstructions; 4) diluents for samples and the standards; 5) a washbuffer; 6) color reagents; 7) stop solution; and 8) a ligand carrier,such as an antibody carrier, for example, a lateral flow device, or amicroplate with bound antibody, or polystyrene beads.

An example of such a kit is a quantitative ELISA (enzyme-linkedimmunosorbent assay) that determines the concentration or concentrationsof the biomarker or biomarker(s) in accordance with methods embodied bythe invention. The principle of the assay is to use the quantitativesandwich enzyme immunoassay technique wherein a monoclonal or polyclonalantibody selective for a biomarker is pre-coated onto a carrier such asa microplate into its wells. The standards and sample are then pipettedinto the wells and any of the biomarker that is present is bound to thisimmobilized antibody. Next, the wells are washed with washing buffer,and an enzyme-linked monoclonal or polyclonal antibody that is specificfor the biomarker is added to the wells. Washing is again performed,then a substrate solution is added to the wells. Color subsequentlydevelops in proportion to the amount of polypeptide of the inventionthat is bound in the first step. The color development is stopped usinga stop solution, and the intensity of the color is measured by amicroplate reader.

The methods of the invention may be carried out using, for example, alateral flow assay. Such lateral flow assays have the potential to be acost-effective, fast, simple, and sensitive method, for instance foron-site screening assays. The lateral flow assay comprises a carrierthat allows a lateral flow to occur wherein either the sample or thedetection reagent is displaced form one location on the carrier toanother. There are many formats of lateral flow assays suitable for usein a method embodied by the invention, and the skilled person willreadily know how to select and optimize a particular format. An exampleof a lateral flow test strip of the invention comprises, for example,the following components:

1. Sample pad—an absorbent pad onto which the test sample is applied.

2. Conjugate or reagent pad—this contains antibodies specific to thetarget analyte conjugated to colored particles (usually colloidal goldparticles, or latex microspheres).

3. Reaction membrane—typically a hydrophobic nitrocellulose or celluloseacetate membrane onto which anti-target analyte antibodies areimmobilized in a line across the membrane as a capture zone or test line(a control zone may also be present, containing antibodies specific forthe conjugate antibodies).

4. Wick or waste reservoir—a further absorbent pad designed to draw thesample across the reaction membrane by capillary action and collect it.

Double antibody sandwich assays—In this format the sample migrates fromthe sample pad through the conjugate pad where any target analytepresent will bind to the conjugate. The sample then continues to migrateacross the membrane until it reaches the capture zone where thetarget/conjugate complex will bind to the immobilized antibodiesproducing a visible line on the membrane. The sample then migratesfurther along the strip until it reaches the control zone, where excessconjugate will bind and produce a second visible line on the membrane.This control line indicates that the sample has migrated across themembrane as intended. Two clear lines on the membrane is a positiveresult. A single line in the control zone is a negative result. Doubleantibody sandwich assays are most suitable for larger analytes, such asbacterial pathogens and viruses, with multiple antigenic sites.Competitive assays are primarily used for testing small molecules anddiffer from the double antibody sandwich format in that the conjugatepad contains antibodies that are already bound to the target analyte, orto an analogue of it. If the target analyte is present in the sample itwill therefore not bind with the conjugate and will remain unlabelled.As the sample migrates along the membrane and reaches the capture zone,an excess of unlabelled analyte will bind to the immobilized antibodiesand block the capture of the conjugate, so that no visible line isproduced. The unbound conjugate will then bind to the antibodies in thecontrol zone producing a visible control line. A single control line onthe membrane is a positive result. Two visible lines in the capture andcontrol zones is a negative result. However, if an excess of unlabelledtarget analyte is not present, a weak line may be produced in thecapture zone, indicating an inconclusive result. Competitive assays aremost suitable for testing for small molecules, such as mycotoxins,unable to bind to more than one antibody simultaneously. There are anumber of variations on lateral flow technology. The capture zone on themembrane may contain immobilized antigens or enzymes—depending on thetarget analyte—rather than antibodies. It is also possible to applymultiple capture zones to create a multiplex test. For example,commercial test strips able to detect both EHEC Shiga toxins ST1 and ST2separately in the same sample have been developed. Lateral flowimmunoassays are simple to use by untrained operators and generallyproduce a result within 15 minutes. They are very stable and robust,have a long shelf life and do not usually require refrigeration. Theyare also relatively inexpensive to produce. These features make themideal for use at the point-of-care and for testing samples in the field,as well as in the laboratory. However, their sensitivity is limitedwithout additional concentration or culture procedures.

Quantitative tests—While most lateral flow immunoassays are only capableof providing a qualitative result, it is possible to obtain some degreeof quantification by measuring the amount of conjugate bound to thecapture zone. This can be done using a dedicated reader to measure theintensity of the colored test line. For example, the Neogen Corporationhas developed the Accuscan® lateral flow reader for use with its rangeof Reveal® assay kits and Charm Sciences also supplies a reader for itsRosa® range of mycotoxin test strips. More sophisticated techniques,such as fluorescent dye labeled conjugates, have also been developed toimprove the quantitative potential of lateral flow assays. Applicationsin the 20 years since the first lateral flow test was launched haveexpanded to include a huge range of different tests that have beendeveloped based on the same technology. The first commercially availablekits were aimed at the clinical diagnostics field, but there are nowproducts with applications in almost every branch of microbiology.Clinical microbiology—lateral flow tests have been developed forbacterial pathogens, respiratory and enteric viruses, intestinalparasites and bacterial toxins.

Many of the lateral flow immunoassay products designed for the clinicalsector were intended to be used at the point-of-care for direct testingof fecal, blood and urine samples and nose and throat swabs, where thesimple operation and speed of the tests is key to their use outside ofthe laboratory. However, the same test strips may also be useful as aquick confirmatory test following laboratory culture of clinicalsamples. Food and agricultural microbiology—test strips are availablefor food borne bacterial pathogens, bacterial and fungal toxins. In thefood microbiology sector, the main applications are more likely to be inthe laboratory, although there are field test kits for mycotoxins ingrain samples. Testing for food borne bacterial pathogens generallyinvolves at least one enrichment stage before the assay strip is used toconfirm the presence or absence of the pathogen. Some manufacturers,such as Dupont®, have developed enrichment media and methodsspecifically designed for use with lateral flow test strips. Test stripsmay also be useful for rapid confirmation of the identity of bacterialisolates from conventional microbiological testing.

A diagnostic system in kit form of the present invention includes, forexample, in an amount sufficient for at least one assay, a polypeptide,antibody composition or monoclonal antibody composition of the presentinvention, as a packaged reagent. Instructions for use of the packagedreagent are also typically included.

A diagnostic system in kit form of the present invention may include,for example, a means for detecting the presence of a biologicalsubstance in a test sample, comprising for example, a lollipop-likeapparatus including a stem integrated with the base and a headintegrated with the stem, for collecting a test sample consisting of,for example, saliva, or a bodily fluid sample from a subject. The stemhead may include a receptor of a sponge like carrier to ensure a highvoid volume to absorb sufficient saliva, or bodily fluid sample. SeeU.S. Pat. No. 7,993,283, incorporated by reference herein in itsentirety.

A diagnostic system in kit form of the present invention may include,for example, a means for combining the test sample with a bufferingsystem (Reagent 1) containing viscosity controllers and stabilizers intoa reaction vessel and mixing the solution. A diagnostic system in kitform of the present invention may include, for example, a means forreading the a parameter of the reaction vessel with sample and buffer,and furter means for combining the test sample and buffer mixture with afluorescence-labeled ligand (Reagent 2) to said biological substance inthe reaction vessel, mixing the solution to produce an assay solution.Furthermore, Reagent 2 may be delivered to the reaction vessel withoutfurther dilution volume of the assay solution.

As used herein, the term “package” refers to a solid matrix or materialsuch as glass, plastic, paper, foil and the like capable of holdingwithin fixed limits a polypeptide, antibody composition or monoclonalantibody composition of the present invention. Thus, for example, apackage can be a glass vial used to contain milligram quantities of acontemplated polypeptide or it can be a microtiter plate well to whichmicrogram quantities of a contemplated polypeptide have been operativelyaffixed, i.e., linked so as to be capable of being immunologically boundby an antibody.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

In preferred embodiments, a diagnostic system of the present inventionfurther includes a label or indicating means capable of signaling theformation of a complex containing a polypeptide or antibody molecule ofthe present invention.

The word “complex” as used herein refers to the product of a specificbinding reaction such as an antibody-antigen or receptor-ligandreaction. Exemplary complexes are immunoreaction products.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. Any label or indicatingmeans can be linked to or incorporated in an expressed protein,polypeptide, or antibody molecule that is part of an antibody ormonoclonal antibody composition of the present invention, or usedseparately, and those atoms or molecules can be used alone or inconjunction with additional reagents such labels are themselveswell-known in clinical diagnostic chemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelproteins methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturing them to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyante (FITC),5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis, etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, or the like. In suchcases where the principal indicating group is an enzyme such as HRP orglucose oxidase, additional reagents are required to visualize the factthat a receptor-ligand complex (immunoreactant) has formed. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2′-azino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

Radioactive elements are also useful labeling agents and are usedillustratively herein.

An exemplary radiolabeling agent is a radioactive element that producesgamma ray emissions. Elements which themselves emit gamma rays, such as¹²⁴, ¹²⁵I, ¹²⁸I, ¹³²I and ⁵¹Cr represent one class of gamma rayemission-producing radioactive element indicating groups. Particularlypreferred is .sup.125I. Another group of useful labeling means are thoseelements such as .sup.11C, .sup.18F, .sup.15O and .sup.13N whichthemselves emit positrons. The positrons so emitted produce gamma raysupon encounters with electrons present in the animal's body. Also usefulis a beta emitter, such ¹¹¹indium or ³H.

The linking of labels, i.e., labeling of, polypeptides and proteins iswell known in the art. For instance, antibody molecules produced by ahybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46(1981). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable. See, forexample, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23(1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No.4,493,795, which are all incorporated herein by reference.

The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A “specific binding agent” is amolecular entity capable of selectively binding a reagent species of thepresent invention or a complex containing such a species, but is notitself a polypeptide or antibody molecule composition of the presentinvention. Exemplary specific binding agents are second antibodymolecules, complement proteins or fragments thereof, S. aureus proteinA, and the like. Preferably the specific binding agent binds the reagentspecies when that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an “ELISA”format to detect, for example, the presence or quantity of cortisol in abody fluid sample such as serum, plasma, or urine, etc. “ELISA” refersto an enzyme-linked immunosorbent assay that employs an antibody orantigen bound to a solid phase and an enzyme-antigen or enzyme-antibodyconjugate to detect and quantify the amount of an antigen or antibodypresent in a sample. A description of the ELISA technique is found inChapter 22 of the 4th Edition of Basic and Clinical Immunology by D. P.Sites et al., published by Lange Medical Publications of Los Altos,Calif. in 1982 and in U.S. Pat. Nos. 3,654,090; 3,850,752; and4,016,043, which are all incorporated herein by reference.

Thus, for example, a polypeptide, antibody molecule composition ormonoclonal antibody molecule composition of the present invention can beaffixed to a solid matrix to form a solid support that comprises apackage in the subject diagnostic systems. The reagent is typicallyaffixed to the solid matrix by adsorption from an aqueous mediumalthough other modes of affixation, well known to those skilled in theart, can be used.

Useful solid matrices are also well known in the art. Such materials arewater insoluble and include cross-linked dextran; agarose; beads ofpolystyrene beads about 1 micron to about 5 millimeters in diameter;polyvinyl chloride, polystyrene, cross-linked polyacrylamide,nitrocellulose- or nylon-based webs such as sheets, strips or paddles;or tubes, plates or the wells of a microtiter plate such as those madefrom polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microtiter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnosticsystems are those customarily utilized in diagnostic systems. Suchmaterials include glass and plastic (e.g., polyethylene, polypropyleneand polycarbonate) bottles, vials, plastic and plastic-foil laminatedenvelopes and the like. In one embodiment a diagnostic system of thepresent invention is useful for assaying for the presence of, forexample, cortisol. Such a system comprises, in kit form, a packagecontaining an antibody to, for example, cortisol.

“Sample” refers to, for example, essentially any source from whichmaterials of interest to be analyzed (e.g., ligands and antiligands,such as antibodies and antigens, and nucleic acids and theircomplements) can be obtained. A sample may be acquired from essentiallyany organism, including animals and plants, as well as cell cultures,recombinant cells and cell components. Samples can be from a biologicaltissue, fluid or specimen and may be obtained from a diseased or healthyorganism. Samples may include, but are not limited to, saliva, sputum,amniotic fluid, blood, blood cells (e.g., white cells), urine, semen,peritoneal fluid, pleural fluid, tissue or fine needle biopsy samples,and tissue homogenates. Samples may also include sections of tissuessuch as frozen sections taken for histological purposes. Typically,samples are taken from a human. However, samples can be obtained fromother mammals also, including by way of example and not limitation,dogs, cats, sheep, cattle, and pigs. The sample may be pretreated asnecessary by dilution in an appropriate buffer solution or concentrated,if desired. Any of a number of standard aqueous buffer solutions,employing one of a variety of buffers, such as phosphate, Tris, or thelike, preferably at physiological pH can be used.

Biological samples can be derived from patients using well knowntechniques such as venipuncture, lumbar puncture, fluid sample such assaliva or urine, or tissue biopsy and the like. When the biologicalmaterial is derived from non-humans, such as commercially relevantlivestock, blood and tissue samples are conveniently obtained fromlivestock processing plants. Alternatively, a biological sample may beobtained from a cell or blood bank where tissue and/or blood are stored,or from an in vitro source, such as a culture of cells. Techniques forestablishing a culture of cells for use as a source for biologicalmaterials are well known to those of skill in the art.

In one embodiment the sample is selected from or is derived from, forexample, microbial products or biological products.

Although the above described example relates to the antigens relating todisease, the immunoassay apparatus could be used, for example, as anallergy test kit, as a test kit for drugs of abuse or for analyzingnon-human derived samples e.g. bovine, porcine, and veterinary tests.

Specific reagents used in the assay device will be selected so as toensure that the particular target analyte is detected as is well knownin the art. The target analyte may be any analyte for example a chemicalreagent which may be organic or inorganic, and which optionallycomprises a hapten, a protein, a polypeptide, a microorganism or anucleic acid sequence.

In particular, the analyte is a hormone such as a fertility hormone likeprogesterone or a stress hormone such as cortisol. However, there is awide range of applications of these types of tests across the entirefield of diagnostics and analysis. Detection of marker proteins orhormones can be diagnostic of certain disease conditions in humans oranimals, and the presence of drugs or drug residues may also be requiredto be detected, for example, in animal husbandry, forensic medicine orin the testing for banned or prohibited drug substances.

Alternatively, the analyte is a chemical reagent, for instance a smallmolecule, which suitably comprises a hapten. Small molecules willgenerally comprise a single recognizable binding site. Typically theywill have a molecular weight of less than 1 kDa.

Where the assay utilizes a labelled binding partner for the analyte andthe analyte is a chemical reagent, the binding partner may comprise anyother reagent which reacts with or otherwise becomes associated with thechemical reagent, either because it forms covalent or ionic bonds withthe reagent, or by the formation of other interactions, such as hydrogenbonding or Van der Waals interactions. For example, where the chemicalreagent is an acid, the binding partner may comprise an alcohol or anamine that forms an ester or amide with the acid under the sorts ofconditions found in the test. Alternatively the binding partner maycomprise a base that forms a salt with the acid. Conversely, where thebinding partner may comprise the acid part of the reactive pair.

Where the analyte is or comprises a hapten or a protein antigen, thebinding partner may comprise an antibody or a binding fragment thereof,which may be monoclonal, polyclonal or recombinant, but preferably ismonoclonal. Where the analyte is a hormone or enzyme, the labelledbinding partner may comprise a labelled receptor for the analyte.

However, where the analyte is itself an immunoglobulin, and inparticular, an antibody, the labelled binding partner may also comprisefor instance, an antigen or recombinant antigen, as well asanti-antibody immunoglobulin such as anti-sera.

Antibodies or binding fragments to small molecules such as haptens, aregenerated by attaching the molecule to an immunogenic reagent andadministering this to an animal such as a mouse or rabbit. Antibodiesare then harvested from the animal in the usual way. Monoclonalantibodies are obtained by fusing spleen cells to hybridoma cells, andselecting those which bind the hapten, using routine procedures.

For example, where the analyte is a biologically active material such asan active agrochemical as discussed above, specific reagents used in theassay device will be selected so as to ensure that the particular targetbiologically active material is detected as is well known in the art.The biologically active material may be any active chemical such as anagrochemical, for example a chemical reagent which may be organic orinorganic, and which optionally comprises a hapten, a protein, apolypeptide, a microorganism or a nucleic acid sequence. Most preferablythe biologically active material is a chemical reagent, for instance asmall molecule, which suitably comprises a hapten. Small molecules willgenerally have a single antibody binding site. Typically they will havea molecular weight of less that 1 kDa.

Antibodies or binding fragments to small molecules such as haptens, aregenerated by attaching the molecule to an immunogenic reagent andadministering this to an animal such as a mouse or rabbit. Antibodiesare then harvested from the animal in the usual way. Monoclonalantibodies are obtained by fusing spleen cells to hybridoma cells, andselecting those which bind the hapten, using routine procedures.

Microarrays

The method of the invention is particularly useful in combination withthe analysis of gene expression profiles. In some embodiments, a geneexpression profile, such as a collection of transcription rates of anumber of genes, is converted to a projected gene expression profile.The projected gene expression profile is a collection of expressionvalues. The conversion is achieved, in some embodiments, by averagingthe transcription rate of the genes. In some other embodiments, otherlinear projection processes may be used.

Microarrays may be prepared and analyzed using methods known in the art.Oligonucleotides may be used as either probes or targets in amicroarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously and to identify geneticvariants, mutations, and single nucleotide polymorphisms. Suchinformation may be used to determine gene function; to understand thegenetic basis of a condition, disease, or disorder; to diagnose acondition, disease, or disorder; and to develop and monitor theactivities of therapeutic agents. (See, e.g., Brennan et al. (1995) U.S.Pat. No. 5,474,796; Schena et al. (1996) Proc. Natd. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon et al. (1995) PCT application WO95/35505; Heller etal. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller et al.(1997); U.S. Pat. No. 5,605,662.) Hybridization probes are also usefulin mapping the naturally occurring genomic sequence. The sequences maybe mapped to a particular chromosome, to a specific region of achromosome, or to artificial chromosome constructions, e.g., humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions, orsingle chromosome DNA libraries.

Using the methods of the invention a skilled artisan can readily selectand prepare probes for a microarray wherein the microarray containsspecific individual probes for less than all the genes in the genome andless than all the genes in the genome. In such embodiments, themicroarray contains one or two or more individual probes, each of whichhybridizes to an expression product (e.g., mRNA, or cDNA or cRNA derivedtherefrom) for a desired number of genes. Thus, for example, changes inthe expression of all or most of the genes in the entire genome of acell or organism can thereby be monitored by use of a surrogate and on asingle microarray by measuring expression of the group of genes that arerepresentative of all or most of the genes of the genome. Suchmicroarrays can be prepared using the selected probes and are thereforepart of the present invention.

Formulations

The compounds and compositions of the invention may be administeredenterally or parenterally. Mixed with pharmaceutically suitableauxiliaries, e.g., as described in the standard reference, Gennaro etal., Remington's Phannaceutical Sciences. The compounds may becompressed into solid dosage units, such as pills, tablets, or beprocessed into capsules or suppositories. By means of pharmaceuticallysuitable liquids the compounds can also be applied in the form of asolution, suspension, emulsion, e.g. for use as an injection preparationor eye drops, or as a spray, e.g. for use as a nasal spray.

For making dosage units, e.g., tablets, the use of conventionaladditives such as fillers, colorants, polymeric binders and the like iscontemplated. In general, any pharmaceutically acceptable additive whichdoes not interfere with the function of the active compounds can beused. Suitable carriers with which the compositions can be administeredinclude lactose, starch, cellulose derivatives and the like, or mixturesthereof, used in suitable amounts.

Dosage Forms

The compositions and compositions of the present invention can beprocessed by agglomeration, air suspension chilling, air suspensiondrying, balling, coacervation, coating, comminution, compression,cryopelletization, encapsulation, extrusion, wet granulation, drygranulation, homogenization, inclusion complexation, lyophilization,melting, microencapsulation, mixing, molding, pan coating, solventdehydration, sonication, spheronization, spray chilling, spraycongealing, spray drying, or other processes known in the art. Thecompositions can be provided in the form of a minicapsule, a capsule, atablet, an implant, a troche, a lozenge (minitablet), a temporary orpermanent suspension, an ovule, a suppository, a wafer, a chewabletablet, a quick or fast dissolving tablet, an effervescent tablet, abuccal or sublingual solid, a granule, a film, a sprinkle, a pellet, abead, a pill, a powder, a triturate, a platelet, a strip or a sachet.Compositions can also be administered as a “dry syrup”, where thefinished dosage form is placed directly on the tongue and swallowed orfollowed with a drink or beverage. These forms are well known in the artand are packaged appropriately. The compositions can be formulated fororal, nasal, buccal, ocular, urethral, transmucosal, vaginal, topical orrectal delivery.

The pharmaceutical composition can be coated with one or more entericcoatings, seal coatings, film coatings, barrier coatings, compresscoatings, fast disintegrating coatings, or enzyme degradable coatings.Multiple coatings can be applied for desired performance. Further, thedosage form can be designed for immediate release, pulsatile release,controlled release, extended release, delayed release, targeted release,synchronized release, or targeted delayed release. Forrelease/absorption control, solid carriers can be made of variouscomponent types and levels or thicknesses of coats, with or without anactive ingredient. Such diverse solid carriers can be blended in adosage form to achieve a desired performance. The definitions of theseterms are known to those skilled in the art. In addition, the dosageform release profile can be affected by a polymeric matrix composition,a coated matrix composition, a multiparticulate composition, a coatedmultiparticulate composition, an ion-exchange resin-based composition,an osmosis-based composition, or a biodegradable polymeric composition.Without wishing to be bound by theory, it is believed that the releasemay be effected through favorable diffusion, dissolution, erosion,ion-exchange, osmosis or combinations thereof.

When formulated as a capsule, the capsule can be a hard or soft gelatincapsule, a starch capsule, or a cellulosic capsule. Although not limitedto capsules, such dosage forms can further be coated with, for example,a seal coating, an enteric coating, an extended release coating, or atargeted delayed release coating. These various coatings are known inthe art, but for clarity, the following brief descriptions are provided:seal coating, or coating with isolation layers: Thin layers of up to 20microns in thickness can be applied for variety of reasons, includingfor particle porosity reduction, to reduce dust, for chemicalprotection, to mask taste, to reduce odor, to minimize gastrointestinalirritation, etc. The isolating effect is proportional to the thicknessof the coating. Water soluble cellulose ethers are preferred for thisapplication. HPMC and ethyl cellulose in combination, or Eudragit E100,may be particularly suitable for taste masking applications. Traditionalenteric coating materials listed elsewhere can also be applied to forman isolating layer.

Extended release coatings are designed to effect delivery over anextended period of time. The extended release coating is apH-independent coating formed of, for example, ethyl cellulose,hydroxypropyl cellulose, methylcellulose, hydroxymethyl cellulose,hydroxyethyl cellulose, acrylic esters, or sodium carboxymethylcellulose. Various extended release dosage forms can be readily designedby one skilled in art to achieve delivery to both the small and largeintestines, to only the small intestine, or to only the large intestine,depending upon the choice of coating materials and/or coating thickness.

Enteric coatings are mixtures of pharmaceutically acceptable excipientswhich are applied to, combined with, mixed with or otherwise added tothe carrier or composition. The coating may be applied to a compressedor molded or extruded tablet, a gelatin capsule, and/or pellets, beads,granules or particles of the carrier or composition. The coating may beapplied through an aqueous dispersion or after dissolving in appropriatesolvent. Additional additives and their levels, and selection of aprimary coating material or materials will depend on the followingproperties: 1. resistance to dissolution and disintegration in thestomach; 2. impermeability to gastric fluids and drug/carrier/enzymewhile in the stomach; 3. ability to dissolve or disintegrate rapidly atthe target intestine site; 4. physical and chemical stability duringstorage; 5. non-toxicity; 6. easy application as a coating (substratefriendly); and 7. economical practicality.

Dosage forms of the compositions of the present invention can also beformulated as enteric coated delayed release oral dosage forms, i.e., asan oral dosage form of a pharmaceutical composition as described hereinwhich utilizes an enteric coating to affect release in the lowergastrointestinal tract. The enteric coated dosage form may be acompressed or molded or extruded tablet/mold (coated or uncoated)containing granules, pellets, beads or particles of the activeingredient and/or other composition components, which are themselvescoated or uncoated. The enteric coated oral dosage form may also be acapsule (coated or uncoated) containing pellets, beads or granules ofthe solid carrier or the composition, which are themselves coated oruncoated.

Delayed release generally refers to the delivery so that the release canbe accomplished at some generally predictable location in the lowerintestinal tract more distal to that which would have been accomplishedif there had been no delayed release alterations. The preferred methodfor delay of release is coating. Any coatings should be applied to asufficient thickness such that the entire coating does not dissolve inthe gastrointestinal fluids at pH below about 5, but does dissolve at pHabout 5 and above. It is expected that any anionic polymer exhibiting apH-dependent solubility profile can be used as an enteric coating in thepractice of the present invention to achieve delivery to the lowergastrointestinal tract. Polymers for use in the present invention areanionic carboxylic polymers.

Shellac, also called purified lac, a refined product obtained from the,resinous secretion of an insect. This coating dissolves in media ofpH>7.

Colorants, detackifiers, surfactants, antifoaming agents, lubricants,stabilizers such as hydroxy propyl cellulose, acid/base may be added tothe coatings besides plasticizers to solubilize or disperse the coatingmaterial, and to improve coating performance and the coated product.

In carrying out the method of the present invention, the combination ofthe invention may be administered to mammalian species, such as dogs,cats, humans, etc. and as such may be incorporated in a conventionalsystemic dosage form, such as a tablet, capsule, elixir or injectable.The above dosage forms will also include the necessary carrier material,excipient, lubricant, buffer, antibacterial, bulking agent (such asmannitol), anti-oxidants (ascorbic acid of sodium bisulfite) or thelike.

The dose administered must be carefully adjusted according to age,weight and condition of the patient, as well as the route ofadministration, dosage form and regimen and the desired result.

The pharmaceutical compositions of the invention may be administered inthe dosage forms in single or divided doses of one to four times daily.It may be advisable to start a patient on a low dose combination andwork up gradually to a high dose combination.

Tablets of various sizes can be prepared, e.g., of about 1 to 2000 mg intotal weight, containing one or both of the active pharmaceuticalingredients, with the remainder being a physiologically acceptablecarrier of other materials according to accepted pharmaceuticalpractice. These tablets can be scored to provide for fractional doses.Gelatin capsules can be similarly formulated.

Liquid formulations can also be prepared by dissolving or suspending oneor the combination of active substances in a conventional liquid vehicleacceptable for pharmaceutical administration so as to provide thedesired dosage in one to four teaspoonful.

Dosage forms can be administered to the patient on a regimen of, forexample, one, two, three, four, five, six, or other doses per day Inorder to more finely regulate the dosage schedule, the active substancesmay be administered separately in individual dosage units at the sametime or carefully coordinated times. Since blood levels are built up andmaintained by a regulated schedule of administration, the same result isachieved by the simultaneous presence of the two substances. Therespective substances can be individually formulated in separate unitdosage forms in a manner similar to that described above.

In formulating the compositions, the active substances, in the amountsdescribed above, may be compounded according to accepted pharmaceuticalpractice with a physiologically acceptable vehicle, carrier, excipient,binder, preservative, stabilizer, flavor, etc., in the particular typeof unit dosage form.

Illustrative of the adjuvants which may be incorporated in tablets arethe following: a binder such as gum tragacanth, acacia, corn starch orgelatin; an excipient such as dicalcium phosphate or cellulose; adisintegrating agent such as corn starch, potato starch, alginic acid orthe like; a lubricant such as stearic acid or magnesium stearate; asweetening agent such as sucrose, aspartame, lactose or saccharin; aflavoring agent such as orange, peppermint, oil of wintergreen orcherry. When the dosage unit form is a capsule, it may contain inaddition to materials of the above type a liquid carrier such as a fattyoil. Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets orcapsules may be coated with shellac, sugar or both. A syrup of elixirmay contain the active compound, water, alcohol or the like as thecarrier, glycerol as solubilizer, sucrose as sweetening agent, methyland propyl parabens as preservatives, a dye and a flavoring such ascherry or orange.

One embodiment of this invention includes methods of treating,preventing, or diagnosing a particular disease or condition byadministering the disclosed nanoparticles, composite nanoparticles,nanosuspension, or nanocapsules to a subject. In many instances, thenanoparticles, composite nanoparticles, or nanocapsules are administeredalone or can be included within a pharmaceutical composition. Aneffective amount of a pharmaceutical composition, generally, is definedas that amount sufficient to ameliorate, reduce, minimize, or limit theextent of the disease or condition. More rigorous definitions may apply,including elimination, eradication, or cure of the disease or condition.

“Nanoparticles” are solid particles of an average particle diameter of,for example, less than about 1 micron (micrometer). One micron is 1,000nanometers (nm).

“Stabilized” nanoparticles are nanoparticles coated with a stabilizingmaterial and having a reduced tendency for aggregation and loss ofdispersion with respect to nanoparticles of the compound of theinvention without a stabilizing coating.

A nano-spray is a spray containing nanoparticles or a spray thatproduces nanoparticles. A nanodispersion is a dispersion containingnanoparticles. A nanosuspension is a suspension containingnanoparticles.

The liquid formulations useful herein may comprise a solvent, solution,suspension, microsuspension, nanosuspension, emulsion, microemulsion,gel or even a melt containing the active component or components. Insome embodiments the nanoparticles, nanofibers, or nanofibrils may be inthe form of, or within or on, granules, powders, suspensions, solutions,dissolvable films, mats, webs, tablets, or releasable forms particularlyreleasable dosage forms. Other particular useful forms are concentratesto which a diluting liquid is added prior to use. The product may alsobe sprayed onto the inner surface of a container to which a liquid isadded later prior to use and the nanoparticles, nanofibers, ornanofibrils, are released into the liquid.

Pharmaceutical compositions of the present invention can includenanoparticles, composite nanoparticles, nanosuspension, or nanocapsulesof the present invention.

In certain non-limiting embodiments, pharmaceutical compositions maycomprise, for example, at least about 0.1% of an active ingredient ornanoparticles, composite nanoparticles, or nanocapsules, for example. Inother embodiments, the an active ingredient or nanoparticles, compositenanoparticles, or nanocapsules may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. In non-limiting examples of aderivable range from the numbers listed herein, a range of about 5mg/kg/body weight to about 100 mg/kg/body weight, about 5microgram/kg/body weight to about 500 milligram/kg/body weight, etc.,can be administered.

The composition may also include various antioxidants to retardoxidation of one or more active ingredient or nanoparticles, compositenanoparticles, nanosuspension, or nanocapsules. The prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

In order to increase the effectiveness of a treatment with thenanoparticles, nanogels, composite nanoparticles, nanosuspension, ornanocapsules of the present invention, it may be desirable to combinethese nanoparticles, composite nanoparticles, or nanocapsules with othertherapies effective in the treatment of a particular disease orcondition.

The formulations as described above may be administered for a prolongedperiod, that is, for as long as the potential for a disease or conditionremains or the symptoms continue.

Packaging/Treatment Kits

The present invention relates to a kit for conveniently and effectivelycarrying out the methods in accordance with the present invention. Suchkits may be suited for the delivery of solid oral forms such as tabletsor capsules. Such a kit may include a number of unit dosages. Such kitscan include a means for containing the dosages oriented in the order oftheir intended use. An example of a means for containing the dosages inthe order of their intended uses is a card. An example of such a kit isa “blister pack”. Blister packs are well known in the packaging industryand are widely used for packaging pharmaceutical unit dosage forms. Ifdesired, the blister can be in the form of a childproof blister, i.e., ablister that is difficult for a child to open, yet can be readily openedby an adult. If desired, a memory aid can be provided, for example inthe form of numbers, letters, or other markings or with a calendarfeature and/or calendar insert, designating the days and the sections ofa day in the treatment schedule in which the dosages can beadministered, such as an AM dose is packaged with a “mid day” and a PMdose.; or an AM dose is packaged with a PM dose. Alternatively, placebodosages, or vitamin or dietary supplements, either in a form similar toor distinct from the pharmaceutical active dosages, can be included.

In one aspect, the package, kit or container comprises a “blisterpackage” (also called a blister pack, or bubble pack). In one aspect,the blister package consists two or more separate compartments: Amdosage of this invention, and PM dosage of this invention, or mid-daydosage of this invention. This blister package is made up of twoseparate material elements: a transparent plastic cavity shaped to theproduct and its blister board backing. These two elements are thenjoined together with a heat sealing process which allows the product tobe hung or displayed. Exemplary types of “blister packages” include:Face seal blister packages, gang run blister packages, mock blisterpackages, interactive blister packages, slide blister packages.

Blister packs, clamshells or trays are forms of packaging used forgoods; thus, the invention provides for blister packs, clamshells ortrays comprising a composition (e.g., a (the multi-ingredientcombination of drugs of the invention) combination of activeingredients) of the invention. Blister packs, clamshells or trays can bedesigned to be non-reclosable, so consumers can tell if a package hasalready opened. They are used to package for sale goods where producttampering is a consideration, such as the pharmaceuticals of theinvention. In one aspect, a blister pack of the invention comprises amoulded PVC base, with raised areas (the “blisters”) to contain thetablets, pills, etc. comprising the combinations of the invention,covered by a foil laminate. Tablets, pills, etc. are removed from thepack either by peeling the foil back or by pushing the blister to forcethe tablet to break the foil. In one aspect, a specialized form of ablister pack is a strip pack.

In one aspect, a blister pack also comprises a method of packaging wherethe compositions comprising combinations of ingredients of the inventionare contained in-between a card and a clear PVC. The PVC can betransparent so the item (pill, tablet, geltab, etc.) can be seen andexamined easily; and in one aspect, can be vacuum-formed around a mouldso it can contain the item snugly and have room to be opened uponpurchase. In one aspect, the card is brightly colored and designeddepending on the item (pill, tablet, geltab, etc.) inside, and the PVCis affixed to the card using pre-formed tabs where the adhesive isplaced. The adhesive can be strong enough so that the pack may hang on apeg, but weak enough so that this way one can tear open the join andaccess the item. Sometimes with large items or multiple enclosed pills,tablets, geltabs, etc., the card has a perforated window for access. Inone aspect, more secure blister packs, e.g., for items such as pills,tablets, geltabs, etc. of the invention are used, and they can compriseof two vacuum-formed PVC sheets meshed together at the edges, with theinformative card inside.

In one aspect, blister packaging comprises at least two components(e.g., is a multi-ingredient combination of drugs of the invention): athermoformed “blister” which houses the product (e.g., a pharmaceuticalcombination of the invention), and then a “blister card” that is aprinted card with an adhesive coating on the front surface. During theassembly process, the blister component, which is most commonly made outof PVC, is attached to the blister card using a blister machine. Thismachine introduces heat to the flange area of the blister whichactivates the glue on the card in that specific area and ultimatelysecures the PVG blister to the printed blister card. The thermoformedPVG blister and the printed blister card can be as small or large.Conventional blister packs can also be sealed (e.g., using an AERGO 8DUO®, SCA Consumer Packaging, Inc., DeKalb, Ill.) using regular heatseal tooling. This alternative aspect, using heat seal tooling, can sealcommon types of thermoformed packaging.

As discussed herein, the products of manufacture of the invention cancomprise the packaging of the therapeutic drug combinations of theinvention, alone or in combination, as “blister packages” or as aplurality of packettes, including as lidded blister packages, liddedblister or blister card or packets, or a shrink wrap.

In one aspect, laminated aluminum foil blister packs are used, e.g., forthe preparation of drugs designed to dissolve immediately in the mouthof a patient. This exemplary process comprises having the drugcombinations of the invention prepared as an aqueous solution(s) whichare dispensed (e.g., by measured dose) into an aluminum (e.g., alufoil)laminated tray portion of a blister pack. This tray is then freeze-driedto form tablets which take the shape of the blister pockets. The alufoillaminate of both the tray and lid fully protects any highly hygroscopicand/or sensitive individual doses. In one aspect, the pack incorporatesa childproof peel open security laminate. In one aspect, the system givetablets an identification mark by embossing a design into the alufoilpocket that is taken up by the tablets when they change from aqueous tosolid state. In one aspect, individual ‘push-through’ blisterpacks/packettes are used, e.g., using hard temper aluminum (e.g.,alufoil) lidding material. In one aspect, hermetically-sealed highbarrier aluminum (e.g., alufoil) laminates are used. In one aspect, anyof the invention's products of manufacture, including kits or blisterpacks, use foil laminations and strip packs, stick packs, sachets andpouches, peelable and non-peelable laminations combining foil, paper,and film for high barrier packaging.

Other means for containing said unit dosages can include bottles andvials, wherein the bottle or vial comprises a memory aid, such as aprinted label for administering said unit dosage or dosages. The labelcan also contain removable reminder stickers for placement on a calendaror dayminder to further help the patient to remember when to take adosage or when a dosage has been taken.

Formulations for Alternate Specific Routes of Administration

The pharmaceutical compositions may be optimized for particular types ofdelivery. For example, pharmaceutical compositions for oral delivery areformulated using pharmaceutically acceptable carriers that are wellknown in the art. The carriers enable the agents in the composition tobe formulated, for example, as a tablet, pill, capsule, solution,suspension, sustained release formulation; powder, liquid or gel fororal ingestion by the subject.

The GCR antagonist may also be delivered in an aerosol spray preparationfrom a pressurized pack, a nebulizer or from a dry powder inhaler.Suitable propellants that can be used in a nebulizer include, forexample, dichlorodifluoro-methane, trichlorofluoromethane,dichlorotetrafluoroethane and carbon dioxide. The dosage can bedetermined by providing a valve to deliver a regulated amount of thecompound in the case of a pressurized aerosol.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. Preferably the compositions are administered by the oral,intranasal or respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulized by use of inert gases. Nebulized solutions may bebreathed directly from the nebulizing device or the nebulizing devicemay be attached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices that deliverthe formulation in an appropriate manner.

Neoplasia Therapies

Any therapy (e.g., therapeutic or prophylactic agent) which is useful,has been used, is currently being used, or may be used for theprevention, treatment and/or management of neoplasia can be used toprevent, treat, and/or manage the patient whose neoplasia and/or cancerstem cells in accordance with the methods, compositions, andcombinations of the invention. Also, neoplasia and/or cancer stem cellmonitoring can be employed in conjunction with any therapy for canceraccording to the instant invention. Therapies (e.g., therapeutic orprophylactic agents) include, but are not limited to, peptides,polypeptides, fusion proteins, nucleic acid molecules, small molecules,mimetic agents, synthetic drugs, inorganic molecules, and organicmolecules. Non-limiting examples of cancer therapies includechemotherapies, radiation therapies, hormonal therapies,anti-angiogenesis therapies, targeted therapies, and/or biologicaltherapies including immunotherapies and surgery. In certain embodiments,a prophylactically and/or therapeutically effective regimen comprisesthe administration of a combination of therapies. In certainembodiments, ORG 34517 can be administered as an agent to treat orprevent neoplasia, alone or in combination with, for example, neoplasiatreating agents. In certain embodiments, RU486 (mifepristone) can beadministered as an agent to treat or prevent neoplasia.

Examples of neoplasia agents or therapies include, but are not limitedto: acivicin; aclarubicin; acodazole hydrochloride; acronine;adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate;aminoglutethimide; amsacrine; anastrozole; anthracyclin; anthramycin;asparaginase; asperlin; azacitidine (Vidaza); azetepa; azotomycin;batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafidedimesylate; bisphosphonates (e.g., pamidronate (Aredria), sodiumclondronate (Bonefos), zoledronic acid (Zometa), alendronate (Fosamax),etidronate, ibandornate, cimadronate, risedromate, and tiludromate);bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine (Ara-C); dacarbazine; dactinomycin;daunorubicin hydrochloride; decitabine (Dacogen); demethylation agents,dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; EphA2 inhibitors; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; histone deacetylase inhibitors(HDAC-Is) hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine;imatinib mesylate (Gleevec, Glivec); interleukin II (includingrecombinant interleukin II, or rIL2), interferon alpha-2a; interferonalpha-2b; interferon alpha-n1; interferon alpha-n3; interferon beta-I a;interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotideacetate; lenalidomide (Revlimid); letrozole; leuprolide acetate;liarozole hydrochloride; lometrexol sodium; lomustine; losoxantronehydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride;anti-CD2 antibodies (e.g., siplizumab (MedImmune Inc.; InternationalPublication No. WO 02/098370, which is incorporated herein by referencein its entirety)); megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mifepristone; mitindomide; mitocarcin; mitocromin;mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantronehydrochloride; mycophenolic acid; nocodazole; nogalamycin; ORG 34517;ormaplatin; oxaliplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; RU486;safingol; safingol hydrochloride; semustine; simtrazene; sparfosatesodium; sparsomycin; spirogermanium hydrochloride; spiromustine;spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin;tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin;teniposide; teroxirone; testolactone; thiamiprine; thioguanine;thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestoloneacetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate;triptorelin; tubulozole hydrochloride; uracil mustard; uredepa;vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate;vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride.

Other examples of cancer therapies include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; dihydrotaxol, dioxamycin; diphenyl spiromustine;docetaxel; docosanol; dolasetron; doxifluridine; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflomithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; HMG CoA reductase inhibitors (e.g.,atorvastatin, cerivastatin, fluvastatin, lescol, lupitor, lovastatin,rosuvastatin, and simvastatin); hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;immunostimulant peptides; insulin-like growth factor-1 receptorinhibitor; interferon agonists; interferons; interleukins; iobenguane;iododoxorubicin; ipomeanol, 4-iroplact; irsogladine; isobengazole;isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinansulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocytealpha interferon; leuprolide+estrogen+progesterone; leuprorelin;levamisole; LFA-3TIP (Biogen, Cambridge, Mass.; InternationalPublication No. WO 93/0686 and U.S. Pat. No. 6,162,432); liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives;palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene;parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfatesodium; pentostatin; pentrozole; perflubron; perfosfamide; perillylalcohol; phenazinomycin; phenylacetate; phosphatase inhibitors;picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetinA; placetin B; plasminogen activator inhibitor; platinum complex;platinum compounds; platinum-triamine complex; porfimer sodium;porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;proteasome inhibitors; protein A-based immune modulator; protein kinaseC inhibitor; protein kinase C inhibitors, microalgal; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors;purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene;raf antagonists; raltitrexed; ramosetron; ras farnesyl proteintransferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptinedemethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RIIretinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginoneB1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim;Sdi 1 mimetics; semustine; senescence derived inhibitor 1; senseoligonucleotides; signal transduction inhibitors; signal transductionmodulators; gamma secretase inhibitors, single chain antigen bindingprotein; sizofuran; sobuzoxane; sodium borocaptate; sodiumphenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stem cell inhibitor; stem-cell division inhibitors;stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactiveintestinal peptide antagonist; suradista; suramin; swainsonine;synthetic glycosaminoglycans; tallimustine; 5-fluorouracil; leucovorin;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;thalidomide; velaresol; veramine; verdins; verteporfin; vinorelbine;vinxaltine; anti-integrin antibodies (e.g., anti-integrin a_(v)b₃antibodies); vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

A non-limiting list of compounds that could be used to target cancerstem cells includes: inhibitors of interleukin-3 receptor (IL-3R) andCD123 (including peptides, peptide-conjugates, antibodies,antibody-conjugates, antibody fragments, and antibodyfragment-conjugates that target IL-3R or CD123); cantharidin;norcantharidin and analogs and derivatives thereof; Notch pathwayinhibitors including gamma secretase inhibitors; sonichedgehog/smoothened pathway inhibitors including cyclopamine and analogsthereof; antibodies to CD96; certain NF-kB/proteasome inhibitorsincluding parthenolide and analogs thereof; certain triterpenesincluding celastrol; certain mTOR inhibitors; compounds and antibodiesthat target the urokinase receptor; sinefungin; certain inosinemonophosphate dehydrogenase (IMPDH) inhibitors; PPAR-alpha andPPAR-gamma agonists and antagonists (including pioglitazone,tesaslitazar, muraglitazar, peliglitazar, lobeglitazone, balaglitazone,ragaglitazar, rosiglitazone, farglitazar, sodelglitazar, reglitazar,naveglitazar, oxeglitazar, metaglidasen, netoglitazone, darglitazone,englitazone, thiazolidinediones, aleglitazar, edaglitazone,rivoglitazone, troglitazone, imiglitazar, and sipoglitazar); telomeraseinhibitors; antibodies to EpCAM (ESA); GSK-3 beta agonists andantagonists (including Lithium, 6-bromoinirubin-3′-oxime (BIO), TDZD8);Wnt pathway inhibitors including antibodies to frizzled or smallmolecules that inhibit disheveled/frizzled or beta catenin; anti-CD20antibodies and conjugates (e.g. Rituxan, Bexxar, Zevalin) for novel usein multiple myeloma or melanoma; anti-CD133 antibody; anti-CD44antibody; antibodies to IL-4; certain differentiation agents such asversnarinone; compounds that target CD33 such as an antibody orbetulinic acid; compounds that target lactadherin such as an antibody;small molecules or antibodies that target CXCR4 or SDF-1; smallmolecules or antibodies that target multi-drug resistance pumps;inhibitors of survivin; inhibitors of XIAP; small molecules that targetBcl-2; antibodies to CLL-1; and furin inhibitors (such ascucurbitacins).

An additional non-limiting list of compounds that could also be used totarget cancer and/or cancer stem cells includes: i) antibodies, antibodyfragments, and proteins that are either naked or conjugated to atherapeutic moiety that target certain cell surface targets on cancerstem cells, or ii) small molecules known in the art including ones thatcan be further optimized (e.g., via chemistry) or identified via acancer stem cell-based screen (e.g., such as one that would determinewhether a compound impairs proliferation or viability of a cancer stemcell through standard methods, the cell surface and intracellulartargets including (not meant to be exhaustive) are: Rex1 (Zfp42), CTGF,Activin A, Wnt, FGF-2, HIF-1, AP-2gamma, Bmi-1, nucleostemin, hiwi,Moz-TIF2, Nanog, beta-arrestin-2, Oct-4, Sox2, stella, GDF3, RUNX3,EBAF, TDGF-1, nodal, ZFPY, PTNE, Evi-1, Pax3, Mcl-1, c-kit, Lex-1, Zfx,lactadherin, aldehyde dehydrogenase, BCRP, telomerase, CD133, Bcl-2,CD26, Gremlin, and FoxC2.

In some embodiments, the therapy(ies) is an immunomodulatory agent.Non-limiting examples of immunomodulatory agents include proteinaceousagents such as cytokines, peptide mimetics, and antibodies (e.g., human,humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2fragments or epitope binding fragments), nucleic acid molecules (e.g.,antisense nucleic acid molecules and triple helices), small molecules,organic compounds, and inorganic compounds. In particular,immunomodulatory agents include, but are not limited to, methotrexate,leflunomide, cyclophosphamide, cytoxan, Immuran, cyclosporine A,minocycline, azathioprine, antibiotics (e.g., FK506 (tacrolimus)),methylprednisolone (MP), corticosteroids, steroids, mycophenolatemofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar,malononitriloamides (e.g., leflunamide), T cell receptor modulators,cytokine receptor modulators, and modulators mast cell modulators. Otherexamples of immunomodulatory agents can be found, e.g., in U.S.Publication No. 2005/0002934 A1 at paragraphs 259-275 which isincorporated herein by reference in its entirety. In one embodiment, theimmunomodulatory agent is a chemotherapeutic agent. In an alternativeembodiment, the immunomodulatory agent is an immunomodulatory agentother than a chemotherapeutic agent. In some embodiments, thetherapy(ies) used in accordance with the invention is not animmunomodulatory agent.

In some embodiments, the therapy(ies) is an anti-angiogenic agent.Non-limiting examples of anti-angiogenic agents include proteins,polypeptides, peptides, fusion proteins, antibodies (e.g., human,humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments,F(ab)₂ fragments, and antigen-binding fragments thereof) such asantibodies that specifically bind to TNF-alpha, nucleic acid molecules(e.g., antisense molecules or triple helices), organic molecules,inorganic molecules, and small molecules that reduce or inhibitangiogenesis. Other examples of anti-angiogenic agents can be found,e.g., in U.S. Publication No. 2005/0002934 A1 at paragraphs 277-282,which is incorporated by reference in its entirety. In otherembodiments, the therapy(ies) is not an anti-angiogenic agent.

In certain embodiments, the therapy(ies) is an alkylating agent, anitrosourea, an antimetabolite, and anthracyclin, a topoisomerase IIinhibitor, or a mitotic inhibitor. Alkylating agents include, but arenot limited to, busulfan, cisplatin, carboplatin, chlorambucil,cyclophosphamide, ifosfamide, decarbazine, mechlorethamine, mephalen,and themozolomide. Nitrosoureas include, but are not limited tocarmustine (BCNU) and lomustine (CCNU). Antimetabolites include but arenot limited to 5-fluorouracil, capecitabine, methotrexate, gemcitabine,cytarabine, and fludarabine. Anthracyclins include but are not limitedto daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone.Topoisomerase II inhibitors include, but are not limited to, topotecan,irinotecan, etopiside (VP-16), and teniposide. Mitotic inhibitorsinclude, but are not limited to taxanes (paclitaxel, docetaxel), and thevinca alkaloids (vinblastine, vincristine, and vinorelbine). In someembodiments of the invention, the therapy(ies) includes theadministration cantharidin or an analog thereof. The invention includesthe use of agents that target cancer stem cells. In certain embodiments,the agent acts alone. In other embodiments, the agent is attacheddirectly or indirectly to another therapeutic moiety. Non-limitingexamples of therapeutic moieties include, but are not limited toalkylating agents, anti-metabolites, plant alkaloids, cytotoxic agents,chemotherapeutic agents (e.g., a steroid, cytosine arabinoside,fluoruracil, methotrexate, aminopterin, mitomycin C, demecolcine,etoposide, mithramycin, calicheamicin, CC-1065, chlorambucil ormeiphalan), radionuclides, therapeutic enzymes, cytokines, toxinsincluding plant-derived toxins, fungus-derived toxins, bacteria-derivedtoxin (e.g., deglycosylated ricin A chain, a ribosome inactivatingprotein, alpha-sarcin, aspergillin, restirictocin, a ribonuclease, adiphtheria toxin, Pseudomonas exotoxin, a bacterial endotoxin or thelipid A moiety of a bacterial endotoxin), growth modulators and RNase.In some embodiments, the agent used is an agent that binds to a marker,e.g., an antigen on a cancer stem cell. In a specific embodiment, theagent binds to an antigen that is expressed at a greater level on cancerstem cells than on normal stem cells. In a specific embodiment, theagent binds specifically to a cancer stem cell antigen that is not anormal stem cell. In other embodiments, the therapy(ies) is an agentthat binds to a marker on cancer stem cells. In one embodiment, theagent that binds to a marker on cancer stem cells is an antibody or anantibody conjugated to a therapeutic moiety or an antibody fragmentconjugated to a therapeutic moiety.

For example, in a specific embodiment, the agent binds specifically tothe IL-3 Receptor (IL-3R). In some embodiments, the agent that binds tothe IL-3R is an antibody or an antibody fragment that is specific forIL-3R. In some embodiments, the antibody or antibody fragment isconjugated either chemically or via recombinant technology to atherapeutic moiety (e.g., a chemotherapeutic agent, a plant-, fungus- orbacteria-derived toxin, a radionuclide) using a linking agent to effecta cell killing response. In certain embodiments, the antibody,antibody-conjugate, antibody fragment, or antibody fragment-conjugatebinds to the .alpha.-subunit of IL-3R (i.e., the CD123 antigen). Inother embodiments, the antibody, antibody-conjugate, antibody fragment,or antibody fragment-conjugate binds to the IL-3R, containing both the.alpha. and .beta. subunits. Methods for preparing antibodies to IL-3Rand mimetics of antibodies to IL-3R are described in U.S. Pat. No.6,733,743 B2, which is incorporated herein by reference in its entirety.

In other embodiments, the agent that binds to a marker on cancer stemcells is a ligand. In some embodiments, the ligand is a cytokine thatbinds to a cytokine receptor on cancer stem cells. In a particularembodiment, the ligand is interleukin-3 (IL-3) which can be conjugatedto a therapeutic moiety that includes a chemotherapeutic agent, aplant-, fungus-, or bacteria-derived toxin, or a radionuclide. TheIL-3-conjugate prophylactic and/or therapeutic therapy or regimen can bein the form of a recombinant fusion protein in embodiments where theconjugate is a toxin and the toxin is a protein, such as diphtheriatoxin. Methods for preparing and isolating an IL-3-diphtheria toxinfusion protein (IL3DT) are described in Frankel et al., “Diphtheriatoxin fused to human interleukin-3 is toxic to blasts from patients withmyeloid leukemias,” Leukemia 14:576 (2000) and Urieto et al., ProteinExpression and Purification 33: 123-133 (2004), the disclosures of whichare incorporated by reference in their entireties.

In certain embodiments, antibodies or fragments thereof that bind to amarker on cancer stem cells are substantially non-immunogenic in thetreated subject. Methods for obtaining non-immunogenic antibodiesinclude, but are not limited to, chimerizing the antibody, humanizingthe antibody, and isolating antibodies from the same species as thesubject receiving the therapy. Antibodies or fragments thereof that bindto markers in cancer stem cells can be produced using techniques knownin the art. See, for example, paragraphs 539-573 of U.S. Publication No.2005/0002934 A1, which is incorporated by reference in its entirety.

In some embodiments, the therapy comprises the use of X-rays, gamma raysand other sources of radiation to destroy cancer stem cells and/orcancer cells. In specific embodiments, the radiation therapy isadministered as external beam radiation or teletherapy, wherein theradiation is directed from a remote source. In other embodiments, theradiation therapy is administered as internal therapy or brachytherapywherein a radioactive source is placed inside the body close to cancerstem cells, cancer cells and/or a tumor mass.

In some embodiments, the therapy used is a proliferation based therapy.Non-limiting examples of such therapies include a chemotherapy andradiation therapy as described supra.

Currently available therapies and their dosages, routes ofadministration and recommended usage are known in the art and have beendescribed in such literature as the Physician's Desk Reference (60thed., 2006).

In a specific embodiment, cycling therapy involves the administration ofa first cancer therapeutic for a period of time, followed by theadministration of a second cancer therapeutic for a period of time,optionally, followed by the administration of a third cancer therapeuticfor a period of time and so forth, and repeating this sequentialadministration, i.e., the cycle in order to reduce the development ofresistance to one of the cancer therapeutics, to avoid or reduce theside effects of one of the cancer therapeutics, and/or to improve theefficacy of the cancer therapeutics.

When two prophylactically and/or therapeutically effective regimens areadministered to a subject concurrently, the term “concurrently” is notlimited to the administration of the cancer therapeutics at exactly thesame time, but rather, it is meant that they are administered to asubject in a sequence and within a time interval such that they can acttogether (e.g., synergistically to provide an increased benefit than ifthey were administered otherwise). For example, the cancer therapeuticsmay be administered at the same time or sequentially in any order atdifferent points in time; however, if not administered at the same time,they should be administered sufficiently close in time so as to providethe desired therapeutic effect, preferably in a synergistic fashion. Thecombination cancer therapeutics can be administered separately, in anyappropriate form and by any suitable route. When the components of thecombination cancer therapeutics are not administered in the samepharmaceutical composition, it is understood that they can beadministered in any order to a subject in need thereof. For example, afirst prophylactically and/or therapeutically effective regimen can beadministered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of the second cancer therapeutic, toa subject in need thereof. In various embodiments, the cancertherapeutics are administered 1 minute apart, 10 minutes apart, 30minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hoursapart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hoursto 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart,10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24hours apart or no more than 48 hours apart. In one embodiment, thecancer therapeutics are administered within the same office visit. Inanother embodiment, the combination cancer therapeutics are administeredat 1 minute to 24 hours apart.

In a specific embodiment, the combination therapies have the samemechanism of action. In another specific embodiment, the combinationtherapies each have a different mechanism of action.

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A method for treating neoplasia in a patient inneed of such treatment, comprising: administering to said patient atleast one therapeutically effective neoplasia-treating therapy, andwherein the GCR antagonist is ORG34517((11β,17β)-11-(1,3-benzodioxol-5-yl)-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one) which has the following formula:

wherein the administration of ORG 34517 treats said animal or human. 2.The method of claim 1, wherein the neoplasia is selected from the groupconsisting of pancreatic cancer, brain cancer, and combinations thereof.3. The method of claim 1, wherein the neoplasia is chemo-resistantER/GR+ breast cancer.
 4. The method of claim 1 wherein saidneoplasia-treating therapy is radiation.
 5. The method of claim 1wherein said neoplasia-treating therapy is a radionuclide.
 6. The methodof claim 1 wherein said neoplasia treating therapy is selected from thegroup consisting of X-rays, gamma rays, and other sources of radiation.7. The method of claim 1 wherein said neoplasia treating therapy isradiation therapy administered as external beam radiation, wherein theradiation is directed from a remote source.
 8. The method of claim 1wherein said neoplasia treating therapy is radiation therapyadministered as brachytherapy wherein a radioactive source is placedinside the body close to cancer stem cells, cancer cells, and/or a tumormass.
 9. The method of claim 1 wherein the method comprisesadministration of a neoplasia treating therapy followed by theadministration of the GCR antagonist ORG34517.
 10. The method of claim 1wherein the method comprises administration of a neoplasia treatingtherapy and the administration of the GCR antagonist ORG34517concurrently.
 11. The method of claim 1 wherein the method comprisesadministration of the GCR antagonist ORG34517 followed by theadministration of a neoplasia treating therapy.
 12. The method of claim1 wherein said neoplasia treating therapy comprises both radiationtherapy and an additional neoplasia treating therapy.
 13. The method ofclaim 1 wherein said additional neoplasia treating therapy is selectedfrom the group consisting of peptides, polypeptides, fusion proteins,nucleic acid molecules, small molecules, mimetic agents, syntheticdrugs, inorganic molecules, organic molecules, hormonal therapies,anti-angiogenesis therapies, targeted therapies, biological therapies,immunotherapy, and surgery.
 14. The method of claim 1, wherein the ORG34517 given systemically through oral or intravenous routes.
 15. Themethod of claim 1, wherein the ORG 34517 is given to accomplish cure orremission of tumor.
 16. The method of claim 1, wherein the ORG 34517 isgiven to accomplish reduction of tumor burden to enhance effectivenessof subsequent surgical resection.